Simian adenoviruses SAdV-43, -45, -46, -47, -48, -49, and -50, and uses thereof

ABSTRACT

A recombinant vector comprises simian adenovirus 43, 45, 46, 47, 48, 49 or 50 sequences and a heterologous gene under the control of regulatory sequences. A cell line which expresses simian adenovirus 43, 45, 46, 47, 48, 49 or 50 gene(s) is also disclosed. Methods of using the vectors and cell lines are provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/548,631, filed Nov. 20, 2014, now U.S. Pat. No. 9,593,346, issuedMar. 14, 2017, which is a continuation of U.S. patent application Ser.No. 13/126,557, filed May 10, 2011, now U.S. Pat. No. 8,940,290, issuedJan. 27, 2015, which is a national stage application under 35 U.S.C. §371 of PCT/US09/62548, filed Oct. 29, 2009, which claims benefit to U.S.Provisional Patent Application Nos. 61/109,958, 61/109,979, 61/110,028,61/109,986, 61/109,957, 61/109,955, and 61/109,997, all filed Oct. 31,2008.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under grant numbers5P30DK047757-15 and 5P01HL059407-10 awarded by the National Institutesof Health. The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

Adenovirus is a double-stranded DNA virus with a genome size of about 36kilobases (kb), which has been widely used for gene transferapplications due to its ability to achieve highly efficient genetransfer in a variety of target tissues and large transgene capacity.Conventionally, E1 genes of adenovirus are deleted and replaced with atransgene cassette consisting of the promoter of choice, cDNA sequenceof the gene of interest and a poly A signal, resulting in a replicationdefective recombinant virus.

Adenoviruses have a characteristic morphology with an icosahedral capsidconsisting of three major proteins, hexon (II), penton base (III) and aknobbed fibre (IV), along with a number of other minor proteins, VI,VIII, IX, IIIa and IVa2 [W. C. Russell, J. Gen Virol., 81:2573-2604(November 2000)]. The virus genome is a linear, double-stranded DNA witha terminal protein attached covalently to the 5′ terminus, which hasinverted terminal repeats (ITRs). The virus DNA is intimately associatedwith the highly basic protein VII and a small peptide pX (formerlytermed mu). Another protein, V, is packaged with this DNA-proteincomplex and provides a structural link to the capsid via protein VI. Thevirus also contains a virus-encoded protease, which is necessary forprocessing of some of the structural proteins to produce matureinfectious virus.

A classification scheme has been developed for the Mastadenovirusfamily, which includes human, simian, bovine, equine, porcine, ovine,canine and opossum adenoviruses. This classification scheme wasdeveloped based on the differing abilities of the adenovirus sequencesin the family to agglutinate red blood cells. The result was sixsubgroups, now referred to as subgroups A, B, C, D, E and F. See, T.Shenk et al., Adenoviridae: The Viruses and their Replication”, Ch. 67,in FIELD'S VIROLOGY, 6^(th) Ed., edited by B. N Fields et al,(Lippincott Raven Publishers, Philadelphia, 1996), p. 111-2112.

Recombinant adenoviruses have been described for delivery ofheterologous molecules to host cells. See, U.S. Pat. No. 6,083,716,which describes the genome of two chimpanzee adenoviruses. Simianadenoviruses, C5, C6 and C7, have been described in U.S. Pat. No.7,247,472 as being useful as vaccine vectors. Other chimpanzeeadenoviruses are described in International Publication No. WO2005/1071093 as being useful for making adenovirus vaccine carriers.

What is needed in the art are effective vectors which avoid the effectof pre-existing immunity to selected adenovirus serotypes in thepopulation.

SUMMARY OF THE INVENTION

Isolated nucleic acid sequences and amino acid sequences of simian(gorilla) adenovirus-43 (SAdV-43), -45 (SAdV-45), -46 (SAdV-46), -47(SAdV-47), simian (cynomolgus) adenovirus-48 (SAdV-48), -49 (SAdV-49),and -50 (SAdV-50), and vectors containing these sequences are providedherein. Also provided are a number of methods for using the vectors andcells containing the SAdV-43, -45, -46, -47, -48, -49 or -50 sequencesdescribed herein.

The methods described herein involve delivering one or more selectedheterologous gene(s) to a mammalian patient by administering a SAdV-43,-45, -46, -47, -48, -49 or -50 vector. Use of the compositions describedherein for vaccination permits presentation of a selected antigen forthe elicitation of protective immune responses. The vectors based onsimian adenovirus 45 may also be used for producing heterologous geneproducts in vitro. Such gene products are themselves useful in a varietyfor a variety of purposes such as are described herein.

These and other embodiments and advantages of the invention aredescribed in more detail below.

DETAILED DESCRIPTION OF THE INVENTION

Novel nucleic acid and amino acid sequences from simian adenoviruses 43,45, 46 and 47, which were isolated from gorilla feces, and from simianadenoviruses 48, 49 and 50, which were isolated from cynomolgus monkeyfeces, are provided. With respect to the Sequence Listing, incorporatedherein by reference, any ‘free text’ under numeric identifier <223> isprovided in a section preceding the Claims captioned SEQUENCE LISTINGFREE TEXT.

Also provided are novel adenovirus vectors and packaging cell lines toproduce those vectors for use in the in vitro production of recombinantproteins or fragments or other reagents. Further provided arecompositions for use in delivering a heterologous molecule fortherapeutic or vaccine purposes. Such therapeutic or vaccinecompositions contain the adenoviral vectors carrying an insertedheterologous molecule. In addition, the novel SAdV-43, -45, -46, -47,-48, -49 and -50 sequences are useful in providing the essential helperfunctions required for production of recombinant adeno-associated viral(AAV) vectors. Thus, helper constructs, methods and cell lines which usethese sequences in such production methods, are provided.

The term “substantial homology” or “substantial similarity,” whenreferring to a nucleic acid or fragment thereof, indicates that, whenoptimally aligned with appropriate nucleotide insertions or deletionswith another nucleic acid (or its complementary strand), there isnucleotide sequence identity in at least about 95 to 99% of the alignedsequences.

The term “substantial homology” or “substantial similarity,” whenreferring to amino acids or fragments thereof, indicates that, whenoptimally aligned with appropriate amino acid insertions or deletionswith another amino acid (or its complementary strand), there is aminoacid sequence identity in at least about 95 to 99% of the alignedsequences.

Preferably, the homology is over full-length sequence, or a proteinthereof, or a fragment thereof which is at least 8 amino acids, or moredesirably, at least 15 amino acids in length. Examples of suitablefragments are described herein.

The term “percent sequence identity” or “identical” in the context ofnucleic acid sequences refers to the residues in the two sequences thatare the same when aligned for maximum correspondence. Where gaps arerequired to align one sequence with another, the degree of scoring iscalculated with respect to the longer sequence without penalty for gaps.Sequences that preserve the functionality of the polynucleotide or apolypeptide encoded thereby are more closely identical. The length ofsequence identity comparison may be over the full-length of the genome(e.g., about 36 kbp), the full-length of an open reading frame of agene, protein, subunit, or enzyme [see, e.g., the tables providing theadenoviral coding regions], or a fragment of at least about 500 to 5000nucleotides, is desired. However, identity among smaller fragments, e.g.of at least about nine nucleotides, usually at least about 20 to 24nucleotides, at least about 28 to 32 nucleotides, at least about 36 ormore nucleotides, may also be desired. Similarly, “percent sequenceidentity” may be readily determined for amino acid sequences, over thefull-length of a protein, or a fragment thereof. Suitably, a fragment isat least about 8 amino acids in length, and may be up to about 700 aminoacids. Examples of suitable fragments are described herein.

Identity is readily determined using such algorithms and computerprograms as are defined herein at default settings. Preferably, suchidentity is over the full length of the protein, enzyme, subunit, orover a fragment of at least about 8 amino acids in length. However,identity may be based upon shorter regions, where suited to the use towhich the identical gene product is being put.

As described herein, alignments are performed using any of a variety ofpublicly or commercially available Multiple Sequence Alignment Programs,such as “Clustal W”, accessible through Web Servers on the internet.Alternatively, Vector NTI® utilities [InVitrogen] are also used. Thereare also a number of algorithms known in the art that can be used tomeasure nucleotide sequence identity, including those contained in theprograms described above. As another example, polynucleotide sequencescan be compared using Fasta, a program in GCG Version 6.1. Fastaprovides alignments and percent sequence identity of the regions of thebest overlap between the query and search sequences. For instance,percent sequence identity between nucleic acid sequences can bedetermined using Fasta with its default parameters (a word size of 6 andthe NOPAM factor for the scoring matrix) as provided in GCG Version 6.1,herein incorporated by reference. Similarly programs are available forperforming amino acid alignments. Generally, these programs are used atdefault settings, although one of skill in the art can alter thesesettings as needed. Alternatively, one of skill in the art can utilizeanother algorithm or computer program that provides at least the levelof identity or alignment as that provided by the referenced algorithmsand programs.

“Recombinant”, as applied to a polynucleotide, means that thepolynucleotide is the product of various combinations of cloning,restriction or ligation steps, and other procedures that result in aconstruct that is distinct from a polynucleotide found in nature. Arecombinant virus is a viral particle comprising a recombinantpolynucleotide. The terms respectively include replicates of theoriginal polynucleotide construct and progeny of the original virusconstruct.

“Heterologous” means derived from a genotypically distinct entity fromthat of the rest of the entity to which it is being compared. Forexample, a polynucleotide introduced by genetic engineering techniquesinto a plasmid or vector derived from a different species is aheterologous polynucleotide. A promoter removed from its native codingsequence and operatively linked to a coding sequence with which it isnot naturally found linked is a heterologous promoter. A site-specificrecombination site that has been cloned into a genome of a virus orviral vector, wherein the genome of the virus does not naturally containit, is a heterologous recombination site. When a polynucleotide with anencoding sequence for a recombinase is used to genetically alter a cellthat does not normally express the recombinase, both the polynucleotideand the recombinase are heterologous to the cell.

As used throughout this specification and the claims, the term“comprise” and its variants including, “comprises”, “comprising”, amongother variants, is inclusive of other components, elements, integers,steps and the like. The term “consists of” or “consisting of” areexclusive of other components, elements, integers, steps and the like.

I. The Simian Adenovirus Sequences

Nucleic acid and amino acid sequences of simian adenovirus-43 (SAdV-43),-45, -46, -47, -48, -49 or -50 are provided in the Sequence Listing,which is incorporated by reference herein. These adenoviruses and theirsequences are isolated from the other material with which they areassociated in nature. SAdV-48, -49, and -50 were isolated fromcynomolgus monkey.

SAdV-43 and SAdV-45 were isolated from gorilla and have been determinedto be in the same subgroup as human subgroup C. SAdV-46 and SAdV-47 wereisolated from gorilla and have been determined to be in the samesubgroup as human subgroup B. Thus, SAdV-43, -45, -46, and -47 andconstructs based on their sequences may be useful for inducing orexpressing cytokines/chemokines, including, IFNα, IL-6, RANTES, and MIP1α.

SAdV-48, -49, and -50 do not readily fit into any of the existingadenovirus families A, B, C, D or E. SAdV-48, -49, and -50 are moreserologically and immunologically distinct from adenoviruses withinthese families, and vectors derived from these adenoviruses are thusanticipated to be less likely to face pre-existing immunity in humanpopulations. Further, SAdV-48, -49, and -50 are anticipated to be usefulfor antigen evaluation in non-human and non-chimpanzee primate species,including, e.g., rhesus and cynomolgus monkeys. This is particularlyuseful where human and chimpanzee models are not available for anyvariety of reasons.

A. Nucleic Acid Sequences

The SAdV-43 nucleic acid sequences provided herein include nucleotides 1to 37189 of SEQ ID NO: 1. The SAdV-45 nucleic acid sequences providedherein include nucleotides 1 to 37152 of SEQ ID NO: 38. The SAdV-46nucleic acid sequences provided herein include nucleotides 1 to 35608 ofSEQ ID NO: 69. The SAdV-47 nucleic acid sequences provided hereininclude nucleotides 1 to 35563 of SEQ ID NO: 99. The SAdV-48 nucleicacid sequences provided herein include nucleotides 1 to 34201 of SEQ IDNO: 129. The SAdV-49 nucleic acid sequences provided herein includenucleotides 1 to 35499 of SEQ ID NO: 157. The SAdV-50 nucleic acidsequences provided herein include nucleotides 1 to 35512 of SEQ ID NO:183. See, Sequence Listing, which is incorporated by reference herein.In one embodiment, the nucleic acid sequences of SAdV-43, -45, -46, -47,-48, -49 and -50 further encompass the strand which is complementary tothe sequences of SEQ ID NOs: 1, 38, 69, 99, 129, 157 and 183,respectively, as well as the RNA and cDNA sequences corresponding to thesequences of the following sequences and their complementary strands. Inanother embodiment, the nucleic acid sequences further encompasssequences which are greater than 98.5% identical, and preferably,greater than about 99% identical, to the Sequence Listing. Also includedin one embodiment, are natural variants and engineered modifications ofthe sequences provided in SEQ ID NOs: 1, 38, 69, 99, 129, 157 and 183and their complementary strands. Such modifications include, forexample, labels that are known in the art, methylation, and substitutionof one or more of the naturally occurring nucleotides with a degeneratenucleotide.

SAdV-43 SAdV-45 SAdV-46 SAdV-47 ORF ORF ORF ORF SEQ ID SEQ ID SEQ ID SEQID Regions NO: 1 NO: 38 NO: 69 NO: 99 ITR  1 . . . 80  1 . . . 74  1 . .. 132  1 . . . 126 E1a 13S Join Join Join Join 12S (556 . . . 1069, (546. . . 1059, (574 . . . 1150, (554 . . . 1130, 9S 1161 . . . 1468) 1149 .. . 1456) 1224 . . . 1449) 1224 . . . 1429) E1b Small 1669 . . . 22201658 . . . 2212 1620 . . . 2162 1600 . . . 2142 T/19K Large 1974 . . .3482 1963 . . . 3474 1925 . . . 3409 1905 . . . 3389 T/55K IX 3576 . . .3974 3567 . . . 3965 3505 . . . 3918 3485 . . . 3898 E2b pTP ComplementComplement Complement Complement (8519 . . . 10474, (8510 . . . 10462,(8461 . . . 10440, (8444 . . . 10423, 13989 . . . 13997) 13979 . . .13987) 13909 . . . 13917) 13895 . . . 13903) Polymerase ComplementComplement Complement Complement (5139 . . . 8717, (5130 . . . 8708,(5090 . . . 8659, (5070 . . . 8642, 13989 . . . 13997) 13979 . . .13987) 13909 . . . 13917) 13895 . . . 13903) IVa2 Complement ComplementComplement Complement (4036 . . . 5366, (4027 . . . 5537, (3987 . . .5320, (3967 . . . 5297, 5645 . . . 5657) 5636 . . . 5648) 5596 . . .5608) 5576 . . . 5588) L1 52/55D 10928 . . . 12151 10919 . . . 1214210921 . . . 12087 10907 . . . 12073 IIIa 12180 . . . 13964 12171 . . .13955 12115 . . . 13875 12101 . . . 13861 L2 Penton 14034 . . . 1598613986 . . . 15977 13962 . . . 15650 13948 . . . 15687 VII 16018 . . .16623 16009 . . . 16614 15657 . . . 16232 15694 . . . 16269 V 16696 . .. 17787 16687 . . . 17757 16278 . . . 17324 16315 . . . 17364 pX 17804 .. . 18049 17774 . . . 18019 17356 . . . 17580 17396 . . . 17617 L3 VI18150 . . . 18893 18120 . . . 18869 17656 . . . 18405 17696 . . . 18442Hexon 19000 . . . 21864 18976 . . . 21834 18528 . . . 21404 18561 . . .21434 Endo-protease 21889 . . . 22518 21859 . . . 22488 21444 . . .22070 21465 . . . 22091 E2a DBP Complement Complement ComplementComplement (22616 . . . 24259) (22586 . . . 24223) (22165 . . . 23721)(22185 . . . 23738) L4 100 kD 24303 . . . 26795 24267 . . . 26762 23752. . . 26244 23769 . . . 26261 33 kD Join Join Join Join homolog (26482 .. . 26824, (26446 . . . 26791, (25943 . . . 26297, (25960 . . . 26314,27015 . . . 27385) 26982 . . . 27352) 26467 . . . 26822) 26484 . . .26842) 22 kD 26482 . . . 27075 26446 . . . 27042 25943 . . . 26554 25960. . . 26574 VIII 27446 . . . 28126 27413 . . . 28093 26895 . . . 2757526915 . . . 27595 E3 12.5K 28130 . . . 28450 28097 . . . 28417 27578 . .. 27892 27598 . . . 27912 CR1* CR1-alpha 28431 . . . 28976 28398 . . .28943 27849 . . . 28292 27869 . . . 28312 7.1K 28963 . . . 29166 28930 .. . 29133 gp19K 29174 . . . 29653 29141 . . . 29620 28280 . . . 2879528300 . . . 28812 CR1- 29701 . . . 30567 29668 . . . 30534 28835 . . .29425 28842 . . . 29477 beta CR1- 30613 . . . 30927 30580 . . . 3089129444 . . . 30202 29499 . . . 30251 gamma RID- 30939 . . . 31208 30903 .. . 31172 30215 . . . 30487 30264 . . . 30536 alpha RID- 31215 . . .31634 31179 . . . 31601 30462 . . . 30896 30511 . . . 30942 beta 14.7K31630 . . . 32013 31597 . . . 31980 30892 . . . 31296 30938 . . . 31342L5 Fiber 32165 . . . 33961 32132 . . . 33934 31535 . . . 32593 31578 . .. 32543 E4 Orf 6/7 Complement Complement Complement Complement (34151 .. . 34426, (34124 . . . 34393, (32633 . . . 32881, (32588 . . . 32836,35129 . . . 35311) 35102 . . . 35284) 33604 . . . 33792) 33550 . . .33747) Orf 6 Complement Complement Complement Complement (34430 . . .35311) (34403 . . . 35284) (32881 . . . 33807) (32836 . . . 33762) Orf 4Complement Complement Complement Complement (35214 . . . 35576) (35187 .. . 35549) (33683 . . . 34063) (33638 . . . 34018) Orf 3 ComplementComplement Complement Complement (35596 . . . 35946) (35569 . . . 35919)(34076 . . . 34426) (34031 . . . 34381) Orf 2 Complement ComplementComplement Complement (35946 . . . 36335) (35919 . . . 36308) (34426 . .. 34812) (34381 . . . 34767) Orf 1 Complement Complement ComplementComplement (36373 . . . 36753) (36346 . . . 36726) (34858 . . . 35229)(34812 . . . 35183) ITR Complement Complement Complement Complement(37110 . . . 37189) (37079 . . . 37152) (35477 . . . 35608) (35438 . . .35563) SAdV-48 SAdV-49 SAdV-50 ORF ORF ORF SEQ ID SEQ ID SEQ ID RegionsNO: 129 NO: 157 NO: 183 ITR  1 . . . 173 1.215  1 . . . 215 E1a 13S JoinJoin Join 12S (491 . . . 1059, (528 . . . 1101, (528 . . . 1101, 9S 1119. . . 1347) 1215 . . . 1441) 1215 . . . 1441) E1b Small 1513 . . . 20641490 . . . 2128 1450 . . . 2128 T/19K Large 1818 . . . 3338 1885 . . .3366 1885 . . . 3366 T/55K IX 3424 . . . 3833 3440 . . . 3886 3440 . . .3886 E2b pTP Complement Complement Complement (8327 . . . 10117, (8433 .. . 10406, (8433 . . . 10406, 13364 . . . 13372) 13592 . . . 13600)13590 . . . 13598) Polymerase Complement Complement Complement (5025 . .. 8522, (5038 . . . 8631, (5038 . . . 8631, 13364 . . . 13372) 13592 . .. 13600) 13590 . . . 13598) IVa2 Complement Complement Complement (3904. . . 5252, (3929 . . . 5265, (3929 . . . 5265, 5531 . . . 5543) 5544 .. . 5556) 5544 . . . 5556) L1 52/55D 10391 . . . 11578 10612 . . . 1174210612 . . . 11742 IIIa 11598 . . . 13334 11764 . . . 13557 11764 . . .13557 L2 Penton 13421 . . . 14932 13651 . . . 15183 13649 . . . 15181VII 14936 . . . 15493 15205 . . . 15759 15203 . . . 15757 V 15547 . . .16611 15809 . . . 16897 15807 . . . 16898 pX 16635 . . . 16853 16918 . .. 17139 16919 . . . 17140 L3 VI 16910 . . . 17677 17198 . . . 1799217199 . . . 17993 Hexon 17776 . . . 20577 18071 . . . 20818 18072 . . .20831 Endo-protease 20615 . . . 21220 20822 . . . 21436 20835 . . .21449 E2a DBP Complement Complement Complement (21280 . . . 22677)(21490 . . . 22968) (21503 . . . 22981) L4 100 kD 22705 . . . 2490023006 . . . 25372 23019 . . . 25385 33 kD Join homolog (24683 . . .24929, 25108 . . . 25397) 22 kD 24683 . . . 25123 25065 . . . 2576325078 . . . 25776 VIII 25454 . . . 26149 25939 . . . 26637 25952 . . .26650 E3 12.5K 26145 . . . 26471 26640 . . . 26963 26653 . . . 26976CR1* 26911 . . . 28335 26924 . . . 28348 CR1-alpha 26419 . . . 275197.1K gp19K CR1- 27619 . . . 28407 beta CR1- gamma RID- 28422 . . . 2869428354 . . . 28623 28367 . . . 28636 alpha RID- 29694 . . . 29008 28533 .. . 28991 28546 . . . 29004 beta 14.7K 28888 . . . 29385 28997 . . .29386 29010 . . . 29399 L5 Fiber 29491 . . . 31200 Fiber 1 Fiber 1 29587. . . 31149 31187 . . . 32440 Fiber 2 Fiber 2 31174 . . . 32427 29600 .. . 31162 E4 Orf 6/7 Complement Complement Complement (31208 . . .31522, (32471 . . . 32701, (32484 . . . 32714, 32239 . . . 32373) 33452. . . 33592) 33465 . . . 33605) Orf 6 Complement Complement Complement(31522 . . . 32373) (32720 . . . 33592) (32733 . . . 33605) Orf 4Complement Complement Complement (32306 . . . 32668) (33528 . . . 33890)(33541 . . . 33903) Orf 3 Complement Complement Complement (32690 . . .33034) (33897 . . . 34244) (33910 . . . 34257) Orf 2 ComplementComplement Complement (33045 . . . 33431) (34258 . . . 34641) (34271 . .. 34654) Orf 1 Complement Complement Complement (33466 . . . 33849)(34670 . . . 35050) (34683 . . . 35063) ITR Complement ComplementComplement (34029 . . . 34201) (35285 . . . 35499) (35298 . . . 35512)*The CR proteins of SAdV-49 and SAdV-50 appear to have the highesthomology to a CRl-beta region. However, since these adenoviruses onlyhave a single CR protein, they have not been so designated, since thealpha, beta, etc. nomenclature typically refers to relative position ofthe open reading frames within the region. It is noted that theseadenoviruses contains two fiber genes, a characteristic they have incommon with certain other monkey adenoviruses, e.g., SAdV-7.

In certain embodiments such as in adenoviral vectors derived from thesesequences, the function of one or both of these regions may beeliminated, e.g., by deletion of the region, destruction of the promoterfor the gene, or another functional deletion. However, in otherembodiments, retention of these regions may be desired.

In one embodiment, fragments of the sequences of SAdV-43, -45, -46, -47,-48, -49 and -50, and their complementary strands, cDNA and RNAcomplementary thereto are provided. Suitable fragments are at least 15nucleotides in length, and encompass functional fragments, i.e.,fragments which are of biological interest. For example, a functionalfragment can express a desired adenoviral product or may be useful inproduction of recombinant viral vectors. Such fragments include the genesequences and fragments listed in the tables herein. The tables providethe transcript regions and open reading frames in the SAdV-43, -45, -46,-47, -48, -49 and -50 sequences. For certain genes, the transcripts andopen reading frames (ORFs) are located on the strand complementary tothat presented in SEQ ID NO: 1. See, e.g., E2b, E4 and E2a. Thecalculated molecular weights of the encoded proteins are also shown.Note that the E1a open reading frames and the E2b open reading frames ofSAdV-43, -45, -46, -47, -48, -49 and -50 contain internal splice sites.These splice sites are noted in the table above.

The SAdV-43, -45, -46, -47, -48, -49 and -50 adenoviral nucleic acidsequences are useful as therapeutic agents and in construction of avariety of vector systems and host cells. As used herein, a vectorincludes any suitable nucleic acid molecule including, naked DNA, aplasmid, a virus, a cosmid, or an episome. These sequences and productsmay be used alone or in combination with other adenoviral sequences orfragments, or in combination with elements from other adenoviral ornon-adenoviral sequences. The SAdV-43, -45, -46, -47, -48, -49 and -50sequences are also useful as antisense delivery vectors, gene therapyvectors, or vaccine vectors. Thus, further provided are nucleic acidmolecules, gene delivery vectors, and host cells which contain theSAdV-43, -45, -46, -47, -48, -49 and -50 sequences.

For example, a nucleic acid molecule containing simian AdV (SAdV)-43,-45, -46, -47, -48, -49 and -50 ITR sequences of the invention. Inanother example, the invention provides a nucleic acid moleculecontaining SAdV-43, -45, -46, -47, -48, -49 and -50 sequences encoding adesired Ad gene product. Still other nucleic acid molecule constructedusing the SAdV-43, -45, -46, -47, -48, -49 and -50 sequences will bereadily apparent to one of skill in the art, in view of the informationprovided herein.

In one embodiment, the simian Ad gene regions identified herein may beused in a variety of vectors for delivery of a heterologous molecule toa cell. For example, vectors are generated for expression of anadenoviral capsid protein (or fragment thereof) for purposes ofgenerating a viral vector in a packaging host cell. Such vectors may bedesigned for expression in trans. Alternatively, such vectors aredesigned to provide cells which stably contain sequences which expressdesired adenoviral functions, e.g., one or more of E1a, E1b, theterminal repeat sequences, E2a, E2b, E4, E4ORF6 region.

In addition, the adenoviral gene sequences and fragments thereof areuseful for providing the helper functions necessary for production ofhelper-dependent viruses (e.g., adenoviral vectors deleted of essentialfunctions, or adeno-associated viruses (AAV)). For such productionmethods, the SAdV-43, -45, -46, -47, -48, -49 and -50 sequences can beutilized in such a method in a manner similar to those described for thehuman Ad. However, due to the differences in sequences between theSAdV-43, -45, -46, -47, -48, -49 and -50 sequences and those of humanAd, the use of the SAdV-43, -45, -46, -47, -48, -49 and -50 sequencesgreatly minimize or eliminate the possibility of homologousrecombination with helper functions in a host cell carrying human Ad E1functions, e.g., 293 cells, which may produce infectious adenoviralcontaminants during rAAV production.

Methods of producing rAAV using adenoviral helper functions have beendescribed at length in the literature with human adenoviral serotypes.See, e.g., U.S. Pat. No. 6,258,595 and the references cited therein.See, also, U.S. Pat. No. 5,871,982 and International Publication Nos. WO99/14354; WO 99/15685; and WO 99/47691. These methods may also be usedin production of non-human serotype AAV, including non-human primate AAVserotypes. The SAdV-43, -45, -46, -47, -48, -49 and -50 sequences whichprovide the necessary helper functions (e.g., E1a, E1b, E2a and/or E4ORF6) can be particularly useful in providing the necessary adenoviralfunction while minimizing or eliminating the possibility ofrecombination with any other adenoviruses present in the rAAV-packagingcell which are typically of human origin. Thus, selected genes or openreading frames of the SAdV-43, -45, -46, -47, -48, -49 and -50 sequencesmay be utilized in these rAAV production methods.

Alternatively, recombinant SAdV-43, -45, -46, -47, -48, -49 and -50vectors may be utilized in these methods. Such recombinant adenoviralsimian vectors may include, e.g., a hybrid simian Ad/AAV in which simianAd sequences flank a rAAV expression cassette composed of, e.g., AAV 3′and/or 5′ ITRs and a transgene under the control of regulatory sequenceswhich control its expression. One of skill in the art will recognizethat still other simian adenoviral vectors and/or SAdV-43, -45, -46,-47, -48, -49 and -50 gene sequences will be useful for production ofrAAV and other viruses dependent upon adenoviral helper.

In still another embodiment, nucleic acid molecules are designed fordelivery and expression of selected adenoviral gene products in a hostcell to achieve a desired physiologic effect. For example, a nucleicacid molecule containing sequences encoding an SAdV-43, -45, -46, -47,-48, -49 or -50 E1a protein may be delivered to a subject for use as acancer therapeutic. Optionally, such a molecule is formulated in alipid-based carrier and preferentially targets cancer cells. Such aformulation may be combined with other cancer therapeutics (e.g.,cisplatin, taxol, or the like). Still other uses for the adenoviralsequences provided herein will be readily apparent to one of skill inthe art.

In addition, one of skill in the art will readily understand that theSAdV-43, -45, -46, -47, -48, -49 and -50 sequences can be readilyadapted for use for a variety of viral and non-viral vector systems forin vitro, ex vivo or in vivo delivery of therapeutic and immunogenicmolecules. For example, the SAdV-43, -45, -46, -47, -48, -49 and -50simian Ad sequences can be utilized in a variety of rAd and non-rAdvector systems. Such vectors systems may include, e.g., plasmids,lentiviruses, retroviruses, poxviruses, vaccinia viruses, andadeno-associated viral systems, among others. Selection of these vectorsystems is not a limitation of the present invention.

Molecules useful for production of the simian and simian-derivedproteins of the invention are also provided. Such molecules which carrypolynucleotides including the SAdV-43, -45, -46, -47, -48, -49 or -50DNA sequences can be in the form of naked DNA, a plasmid, a virus or anyother genetic element.

B. Adenoviral Proteins

SAdV-43, -45, -46, -47, -48, -49 and -50 adenoviruses, such as proteins,enzymes, and fragments thereof, which are encoded by the adenoviralnucleic acids described herein are provided. Further encompassed areSAdV-43, -45, -46, -47, -48, -49 and -50 proteins, enzymes, andfragments thereof, having the amino acid sequences encoded by thesenucleic acid sequences which are generated by other methods. Suchproteins include those encoded by the open reading frames identified inthe table above, the proteins in the Table below (also shown in theSequence Listing) and fragments thereof of the proteins andpolypeptides.

SAdV-43 SAdV-45 SAdV-46 SAdV-47 SAdV-48 SAdV-49 SAdV-50 Regions SEQ IDNO: SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO:E1a 13S 29 66 96 126 154 182 208 12S 9S E1b Small T/19K 2 39 70 100 130158 184 Large T/55K 24 61 91 121 148 177 203 IX 3 40 71 101 131 159 185L1 52/55 kD 4 41 72 102 132 160 186 IIIa 5 42 73 103 133 161 187 L2Penton 6 43 74 104 134 162 188 VII 7 44 75 105 135 163 189 V 8 45 76 106136 164 190 pX 9 46 77 107 137 165 191 L3 VI 10 47 78 108 138 166 192Hexon 11 48 79 109 139 167 193 Endoprotease 12 49 80 110 140 168 194 L4100 kD 13 50 81 111 141 169 195 33 kD 31 68 98 128 156 homolog 22 kD 2562 92 122 150 178 204 VIII 14 51 82 112 142 170 196 E3 12.5K 15 52 83113 151 171 197 CR1 179 205 CR1-alpha 26 63 93 123 143 7.1K 16 53 gp19K17 54 84 114 CR1-beta 18 55 85 115 144 CR1-gamma 19 56 86 116 RID-alpha20 57 87 117 145 172 198 RID-beta 21 58 94 124 152 180 206 14.7K 27 6488 118 146 173 199 L5 Fiber 22 59 89 119 147 Fiber 1-174 Fiber 1-201Fiber 2-175 Fiber 2-200

Thus, in one aspect, unique simian adenoviral 43, -45, -46, -47, -48,-49 and -50 proteins which are substantially pure, i.e., are free ofother viral and proteinaceous proteins are provided. Preferably, theseproteins are at least 10% homogeneous, more preferably 60% homogeneous,and most preferably 95% homogeneous.

In one embodiment, unique simian-derived capsid proteins are provided.As used herein, a simian-derived capsid protein includes any adenoviralcapsid protein that contains a SAdV-43, -45, -46, -47, -48, -49 or -50capsid protein or a fragment thereof, as defined above, including,without limitation, chimeric capsid proteins, fusion proteins,artificial capsid proteins, synthetic capsid proteins, and recombinantcapsid proteins, without limitation to means of generating theseproteins.

Suitably, these simian-derived capsid proteins contain one or moreSAdV-43, -45, -46, -47, -48, -49 or -50 regions or fragments thereof(e.g., a hexon, penton, fiber, or fragment thereof) in combination withcapsid regions or fragments thereof of different adenoviral serotypes,or modified simian capsid proteins or fragments, as described herein. A“modification of a capsid protein associated with altered tropism” asused herein includes an altered capsid protein, i.e., a penton, hexon orfiber protein region, or fragment thereof, such as the knob domain ofthe fiber region, or a polynucleotide encoding same, such thatspecificity is altered. The simian-derived capsid may be constructedwith one or more of the simian SAdV-43, -45, -46, -47, -48, -49 or -50or another Ad serotype which may be of human or non-human origin. SuchAd may be obtained from a variety of sources including the ATCC,commercial and academic sources, or the sequences of the Ad may beobtained from GenBank or other suitable sources.

The amino acid sequences of the penton proteins of SAdV-43 [SEQ ID NO:6], SAdV-45 [SEQ ID NO: 43], SAdV-46 [SEQ ID NO: 74], SAdV-47 [SEQ IDNO: 104], SAdV-48 [SEQ ID NO: 134], SAdV-49 [SEQ ID NO: 162], andSAdV-50 [SEQ ID NO: 188] are provided. Suitably, the penton protein, orunique fragments thereof, may be utilized for a variety of purposes.Examples of suitable fragments include the penton having N-terminaland/or C-terminal truncations of about 50, 100, 150, or 200 amino acids,based upon the amino acid numbering provided above and in SEQ ID NO: 6,43, 74, 104, 134, 162 or 188. Other suitable fragments include shorterinternal, C-terminal, or N-terminal fragments. Further, the pentonprotein may be modified for a variety of purposes known to those ofskill in the art.

The amino acid sequences of the hexon proteins of SAdV-43 [SEQ ID NO:11], SAdV-45 [SEQ ID NO: 48], SAdV-46 [SEQ ID NO: 79], SAdV-47 [SEQ IDNO: 109], SAdV-48 [SEQ ID NO: 139], SAdV-49 [SEQ ID NO: 167], andSAdV-50 [SEQ ID NO: 193] are also provided. Suitably, the hexon protein,or unique fragments thereof, may be utilized for a variety of purposes.Examples of suitable fragments include the hexon having N-terminaland/or C-terminal truncations of about 50, 100, 150, 200, 300, 400, or500 amino acids, based upon the amino acid numbering provided above andin SEQ ID NO: 11, 48, 79, 109, 139, 167 and 193. Other suitablefragments include shorter internal, C-terminal, or N-terminal fragments.For example, one suitable fragment the loop region (domain) of the hexonprotein, designated DE1 and FG1, or a hypervariable region thereof Suchfragments include the regions spanning amino acid residues about 125 to443; about 138 to 441, or smaller fragments, such as those spanningabout residue 138 to residue 163; about 170 to about 176; about 195 toabout 203; about 233 to about 246; about 253 to about 264; about 287 toabout 297; and about 404 to about 430 of the simian hexon proteins, withreference to SEQ ID NO: 11, 48, 79, 109, 139, 167 or 193. Other suitablefragments may be readily identified by one of skill in the art. Further,the hexon protein may be modified for a variety of purposes known tothose of skill in the art. Because the hexon protein is the determinantfor serotype of an adenovirus, such artificial hexon proteins wouldresult in adenoviruses having artificial serotypes. Other artificialcapsid proteins can also be constructed using the 11, 48, 79, 109, 139,167 and/or 193 penton sequences and/or fiber sequences and/or fragmentsthereof.

In one embodiment, an adenovirus having an altered hexon proteinutilizing the sequences of a SAdV-43, -45, -46, -47, -48, -49 or -50hexon protein may be generated. One suitable method for altering hexonproteins is described in U.S. Pat. No. 5,922,315, which is incorporatedby reference. In this method, at least one loop region of the adenovirushexon is changed with at least one loop region of another adenovirusserotype. Thus, at least one loop region of such an altered adenovirushexon protein is a simian Ad hexon loop region of SAdV-43, -45, -46,-47, -48, -49 or -50. In one embodiment, a loop region of the SAdV-43,-45, -46, -47, -48, -49 or -50 hexon protein is replaced by a loopregion from another adenovirus serotype. In another embodiment, the loopregion of the SAdV-43, -45, -46, -47, -48, -49 or -50 hexon is used toreplace a loop region from another adenovirus serotype. Suitableadenovirus serotypes may be readily selected from among human andnon-human serotypes, as described herein. The selection of a suitableserotype is not a limitation of the present invention. Still other usesfor the SAdV-43, -45, -46, -47, -48, -49 and -50 hexon protein sequenceswill be readily apparent to those of skill in the art.

The amino acid sequences of the fiber proteins of SAdV-43 [SEQ ID NO:22], SAdV-45 [SEQ ID NO: 59], SAdV-46 [SEQ ID NO: 89], SAdV-47 [SEQ IDNO: 119] and SAdV-48 [SEQ ID NO: 147] are provided. SAdV-49 and SAdV-50each have two fiber proteins. Fiber proteins 1 and 2 of SAdV-49 have theamino acid sequence of SEQ ID NOs: 174 and 175, respectively. Fiberproteins 1 and 2 of SAdV-50 have the amino acid sequence of SEQ ID NOs:201 and 200, respectively. Suitably, these fiber proteins, or uniquefragments thereof, may be utilized for a variety of purposes. Onesuitable fragment is the fiber knob. Examples of other suitablefragments include the fiber having N-terminal and/or C-terminaltruncations of about 50, 100, 150, or 200 amino acids, based upon theamino acid numbering provided in SEQ ID NOs: 22, 59, 89, 119, 147, 174,175, 200 and 201. Still other suitable fragments include internalfragments. Further, the fiber protein may be modified using a variety oftechniques known to those of skill in the art.

Unique fragments of the proteins of SAdV-43, -45, -46, -47, -48, -49 and-50 are at least 8, at least 15, or at least 20 amino acids in length.However, fragments of other desired lengths can be readily utilized. Inaddition, modifications as may be introduced to enhance yield and/orexpression of a SAdV-43, -45, -46, -47, -48, -49 or -50 gene product,e.g., construction of a fusion molecule in which all or a fragment ofthe SAdV-43, -45, -46, -47, -48, -49 or -50 gene product is fused(either directly or via a linker) with a fusion partner to enhance areprovided herein. Other suitable modifications include, withoutlimitation, truncation of a coding region (e.g., a protein or enzyme) toeliminate a pre- or pro-protein ordinarily cleaved and to provide themature protein or enzyme and/or mutation of a coding region to provide asecretable gene product. Still other modifications will be readilyapparent to one of skill in the art. Further encompassed are proteinshaving at least about 99% identity to the SAdV-43, -45, -46, -47, -48,-49 or -50 proteins provided herein.

As described herein, vectors containing the adenoviral capsid proteinsof SAdV-43, -45, -46, -47, -48, -49 or -50 are particularly well suitedfor use in applications in which the neutralizing antibodies diminishthe effectiveness of other Ad serotype based vectors, as well as otherviral vectors. The rAd vectors are particularly advantageous inreadministration for repeat gene therapy or for boosting immune response(vaccine titers).

Under certain circumstances, it may be desirable to use one or more ofthe SAdV-43, -45, -46, -47, -48, -49 or -50 gene products (e.g., acapsid protein or a fragment thereof) to generate an antibody. The term“an antibody,” as used herein, refers to an immunoglobulin moleculewhich is able to specifically bind to an epitope. The antibodies mayexist in a variety of forms including, for example, high affinitypolyclonal antibodies, monoclonal antibodies, synthetic antibodies,chimeric antibodies, recombinant antibodies and humanized antibodies.Such antibodies originate from immunoglobulin classes IgG, IgM, IgA, IgDand IgE.

Such antibodies may be generated using any of a number of methods knowin the art. Suitable antibodies may be generated by well-knownconventional techniques, e.g., Kohler and Milstein and the many knownmodifications thereof. Similarly desirable high titer antibodies aregenerated by applying known recombinant techniques to the monoclonal orpolyclonal antibodies developed to these antigens [see, e.g., PCT PatentApplication No. PCT/GB85/00392; British Patent Application PublicationNo. GB2188638A; Amit et al., 1986 Science, 233:747-753; Queen et al.,1989 Proc. Nat'l. Acad. Sci. USA, 86:10029-10033; PCT Patent ApplicationNo. PCT/WO9007861; and Riechmann et al., Nature, 332:323-327 (1988);Huse et al, 1988a Science, 246:1275-1281]. Alternatively, antibodies canbe produced by manipulating the complementarity determining regions ofanimal or human antibodies to SAdV-43, -45, -46, -47, -48, -49 or -50,or a protein or other fragment thereof. See, e.g., E. Mark and Padlin,“Humanization of Monoclonal Antibodies”, Chapter 4, The Handbook ofExperimental Pharmacology, Vol. 113, The Pharmacology of MonoclonalAntibodies, Springer-Verlag (June, 1994); Harlow et al., 1999, UsingAntibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press,NY; Harlow et al., 1989, Antibodies: A Laboratory Manual, Cold SpringHarbor, New York; Houston et al., 1988, Proc. Natl. Acad. Sci. USA85:5879-5883; and Bird et al., 1988, Science 242:423-426.

Further provided are anti-idiotype antibodies (Ab2) andanti-anti-idiotype antibodies (Ab3). See, e.g., M. Wettendorff et al.,“Modulation of anti-tumor immunity by anti-idiotypic antibodies.” InIdiotypic Network and Diseases, ed. by J. Cerny and J. Hiernaux, 1990 J.Am. Soc. Microbiol., Washington D.C.: pp. 203-229]. These anti-idiotypeand anti-anti-idiotype antibodies are produced using techniques wellknown to those of skill in the art. These antibodies may be used for avariety of purposes, including diagnostic and clinical methods and kits.

Under certain circumstances, it may be desirable to introduce adetectable label or a tag onto a SAdV-43, -45, -46, -47, -48, -49 or -50gene product, antibody or other construct. As used herein, a detectablelabel is a molecule which is capable, alone or upon interaction withanother molecule, of providing a detectable signal. Most desirably, thelabel is detectable visually, e.g. by fluorescence, for ready use inimmunohistochemical analyses or immunofluorescent microscopy. Forexample, suitable labels include fluorescein isothiocyanate (FITC),phycoerythrin (PE), allophycocyanin (APC), coriphosphine-O (CPO) ortandem dyes, PE-cyanin-5 (PC5), and PE-Texas Red (ECD). All of thesefluorescent dyes are commercially available, and their uses known to theart. Other useful labels include a colloidal gold label. Still otheruseful labels include radioactive compounds or elements. Additionally,labels include a variety of enzyme systems that operate to reveal acolorimetric signal in an assay, e.g., glucose oxidase (which usesglucose as a substrate) releases peroxide as a product which in thepresence of peroxidase and a hydrogen donor such as tetramethylbenzidine (TMB) produces an oxidized TMB that is seen as a blue color.Other examples include horseradish peroxidase (HRP), alkalinephosphatase (AP), and hexokinase in conjunction with glucose-6-phosphatedehydrogenase which reacts with ATP, glucose, and NAD+ to yield, amongother products, NADH that is detected as increased absorbance at 340 nmwavelength.

Other label systems that are utilized in the methods described hereinare detectable by other means, e.g., colored latex microparticles [BangsLaboratories, Indiana] in which a dye is embedded are used in place ofenzymes to form conjugates with the target sequences provide a visualsignal indicative of the presence of the resulting complex in applicableassays.

Methods for coupling or associating the label with a desired moleculeare similarly conventional and known to those of skill in the art. Knownmethods of label attachment are described [see, for example, Handbook ofFluorescent probes and Research Chemicals, 6th Ed., R. P. M. Haugland,Molecular Probes, Inc., Eugene, Ore., 1996; Pierce Catalog and Handbook,Life Science and Analytical Research Products, Pierce Chemical Company,Rockford, IL, 1994/1995]. Thus, selection of the label and couplingmethods do not limit this invention.

The sequences, proteins, and fragments of SAdV-43, -45, -46, -47, -48,-49 and -50 may be produced by any suitable means, including recombinantproduction, chemical synthesis, or other synthetic means. Suitableproduction techniques are well known to those of skill in the art. See,e.g., Sambrook et al, Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Press (Cold Spring Harbor, N.Y.). Alternatively, peptidescan also be synthesized by the well known solid phase peptide synthesismethods (Merrifield, J. Am. Chem. Soc., 85:2149 (1962); Stewart andYoung, Solid Phase Peptide Synthesis (Freeman, San Francisco, 1969) pp.27-62). These and other suitable production methods are within theknowledge of those of skill in the art and are not a limitation of thepresent invention.

In addition, one of skill in the art will readily understand that theSAdV-43, -45, -46, -47, -48, -49 and -50 sequences can be readilyadapted for use for a variety of viral and non-viral vector systems forin vitro, ex vivo or in vivo delivery of therapeutic and immunogenicmolecules. For example, in one embodiment, the simian Ad capsid proteinsand other simian adenovirus proteins described herein are used fornon-viral, protein-based delivery of genes, proteins, and otherdesirable diagnostic, therapeutic and immunogenic molecules. In one suchembodiment, a protein of SAdV-43, -45, -46, -47, -48, -49 or -50 islinked, directly or indirectly, to a molecule for targeting to cellswith a receptor for adenoviruses. Preferably, a capsid protein such as ahexon, penton, fiber or a fragment thereof having a ligand for a cellsurface receptor is selected for such targeting. Suitable molecules fordelivery are selected from among the therapeutic molecules describedherein and their gene products. A variety of linkers including, lipids,polyLys, and the like may be utilized as linkers. For example, thesimian penton protein may be readily utilized for such a purpose byproduction of a fusion protein using the simian penton sequences in amanner analogous to that described in Medina-Kauwe LK, et al, Gene Ther.2001 May; 8(10):795-803 and Medina-Kauwe LK, et al, Gene Ther. 2001 Dec;8(23): 1753-1761. Alternatively, the amino acid sequences of simian Adprotein IX may be utilized for targeting vectors to a cell surfacereceptor, as described in US Patent Publication No. 20010047081.Suitable ligands include a CD40 antigen, an RGD-containing orpolylysine-containing sequence, and the like. Still other simian Adproteins, including, e.g., the hexon protein and/or the fiber protein,may be used for used for these and similar purposes.

Still other SAdV-43, -45, -46, -47, -48, -49 or -50 adenoviral proteinsmay be used as alone, or in combination with other adenoviral protein,for a variety of purposes which will be readily apparent to one of skillin the art. In addition, still other uses for the SAdV-43, -45, -46,-47, -48, -49 and -50 adenoviral proteins will be readily apparent toone of skill in the art.

II. Recombinant Adenoviral Vectors

The compositions described herein include vectors that deliver aheterologous molecule to cells, either for therapeutic or vaccinepurposes. As used herein, a vector may include any genetic elementincluding, without limitation, naked DNA, a phage, transposon, cosmid,episome, plasmid, or a virus. Such vectors contain simian adenovirus DNAof SAdV-43, -45, -46, -47, -48, -49 and/or -50 and a minigene. By“minigene” is meant the combination of a selected heterologous gene andthe other regulatory elements necessary to drive translation,transcription and/or expression of the gene product in a host cell.

Typically, a SAdV-derived adenoviral vector is designed such that theminigene is located in a nucleic acid molecule which contains otheradenoviral sequences in the region native to a selected adenoviral gene.The minigene may be inserted into an existing gene region to disrupt thefunction of that region, if desired. Alternatively, the minigene may beinserted into the site of a partially or fully deleted adenoviral gene.For example, the minigene may be located in the site of such as the siteof a functional E1 deletion or functional E3 deletion, among others thatmay be selected. The term “functionally deleted” or “functionaldeletion” means that a sufficient amount of the gene region is removedor otherwise damaged, e.g., by mutation or modification, so that thegene region is no longer capable of producing functional products ofgene expression. If desired, the entire gene region may be removed.Other suitable sites for gene disruption or deletion are discussedelsewhere in the application.

For example, for a production vector useful for generation of arecombinant virus, the vector may contain the minigene and either the 5′end of the adenoviral genome or the 3′ end of the adenoviral genome, orboth the 5′ and 3′ ends of the adenoviral genome. The 5′ end of theadenoviral genome contains the 5′ cis-elements necessary for packagingand replication; i.e., the 5′ inverted terminal repeat (ITR) sequences(which function as origins of replication) and the native 5′ packagingenhancer domains (that contain sequences necessary for packaging linearAd genomes and enhancer elements for the E1 promoter). The 3′ end of theadenoviral genome includes the 3′ cis-elements (including the ITRs)necessary for packaging and encapsidation. Suitably, a recombinantadenovirus contains both 5′ and 3′ adenoviral cis-elements and theminigene is located between the 5′ and 3′ adenoviral sequences. ASAdV-43, -45, -46, -47, -48, -49 or -50 based adenoviral vector may alsocontain additional adenoviral sequences from these or other sources.

Suitably, these SAdV-43, -45, -46, -47, -48, -49 or -50 based adenoviralvectors contain one or more adenoviral elements derived from theadenoviral genome of SAdV-43, -45, -46, -47, -48, -49 or -50,respectively. In one embodiment, the vectors contain adenoviral ITRsfrom SAdV-43, -45, -46, -47, -48, -49 or -50 and additional adenoviralsequences from the same adenoviral serotype. In another embodiment, thevectors contain adenoviral sequences that are derived from a differentadenoviral serotype than that which provides the ITRs.

As defined herein, a pseudotyped adenovirus refers to an adenovirus inwhich the capsid protein of the adenovirus is from a differentadenovirus than the adenovirus which provides the ITRs.

Further, chimeric or hybrid adenoviruses may be constructed using theadenoviruses described herein using techniques known to those of skillin the art. See, e.g., U.S. Pat. No. 7,291,498.

The selection of the adenoviral source of the ITRs and the source of anyother adenoviral sequences present in vector is not a limitation of thepresent embodiment. A variety of adenovirus strains are available fromthe American Type Culture Collection, Manassas, Va., or available byrequest from a variety of commercial and institutional sources. Further,the sequences of many such strains are available from a variety ofdatabases including, e.g., PubMed and GenBank. Homologous adenovirusvectors prepared from other simian or from human adenoviruses aredescribed in the published literature [see, for example, U.S. Pat. No.5,240,846]. The DNA sequences of a number of adenovirus types areavailable from GenBank, including type Ad5 [GenBank Accession No.M73260]. The adenovirus sequences may be obtained from any knownadenovirus serotype, such as serotypes 2, 3, 4, 7, 12 and 40, andfurther including any of the presently identified human types. Similarlyadenoviruses known to infect non-human animals (e.g., simians) may alsobe employed in the vector constructs. See, e.g., U.S. Pat. No.6,083,716.

The viral sequences, helper viruses (if needed), and recombinant viralparticles, and other vector components and sequences employed in theconstruction of the vectors described herein are obtained as describedabove. The DNA sequences of SAdV-43, -45, -46, -47, -48, -49 and/or -50are employed to construct vectors and cell lines useful in thepreparation of such vectors.

Modifications of the nucleic acid sequences forming the SAdV-43, -45,-46, -47, -48, -49 and/or -50 vectors, including sequence deletions,insertions, and other mutations may be generated using standardmolecular biological techniques and are within the scope of thisembodiment.

A. The “Minigene”

The methods employed for the selection of the transgene, the cloning andconstruction of the “minigene” and its insertion into the viral vectorare within the skill in the art given the teachings provided herein.

1. The Transgene

The transgene is a nucleic acid sequence, heterologous to the vectorsequences flanking the transgene, which encodes a polypeptide, protein,or other product, of interest. The nucleic acid coding sequence isoperatively linked to regulatory components in a manner which permitstransgene transcription, translation, and/or expression in a host cell.

The composition of the transgene sequence will depend upon the use towhich the resulting vector will be put. For example, one type oftransgene sequence includes a reporter sequence, which upon expressionproduces a detectable signal. Such reporter sequences include, withoutlimitation, DNA sequences encoding β-lactamase, β-galactosidase (LacZ),alkaline phosphatase, thymidine kinase, green fluorescent protein (GFP),chloramphenicol acetyltransferase (CAT), luciferase, membrane boundproteins including, for example, CD2, CD4, CD8, the influenzahemagglutinin protein, and others well known in the art, to which highaffinity antibodies directed thereto exist or can be produced byconventional means, and fusion proteins comprising a membrane boundprotein appropriately fused to an antigen tag domain from, among others,hemagglutinin or Myc. These coding sequences, when associated withregulatory elements which drive their expression, provide signalsdetectable by conventional means, including enzymatic, radiographic,colorimetric, fluorescence or other spectrographic assays, fluorescentactivating cell sorting assays and immunological assays, includingenzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA) andimmunohistochemistry. For example, where the marker sequence is the LacZgene, the presence of the vector carrying the signal is detected byassays for beta-galactosidase activity. Where the transgene is GFP orluciferase, the vector carrying the signal may be measured visually bycolor or light production in a luminometer.

In one embodiment, the transgene is a non-marker sequence encoding aproduct which is useful in biology and medicine, such as proteins,peptides, RNA, enzymes, or catalytic RNAs. Desirable RNA moleculesinclude tRNA, dsRNA, ribosomal RNA, catalytic RNAs, and antisense RNAs.One example of a useful RNA sequence is a sequence which extinguishesexpression of a targeted nucleic acid sequence in the treated animal.

The transgene may be used for treatment, e.g., of genetic deficiencies,as a cancer therapeutic or vaccine, for induction of an immune response,and/or for prophylactic vaccine purposes. As used herein, induction ofan immune response refers to the ability of a molecule (e.g., a geneproduct) to induce a T cell and/or a humoral immune response to themolecule. The use of multiple transgenes is contemplated, e.g., tocorrect or ameliorate a condition caused by a multi-subunit protein. Incertain situations, a different transgene may be used to encode eachsubunit of a protein, or to encode different peptides or proteins. Thisis desirable when the size of the DNA encoding the protein subunit islarge, e.g., for an immunoglobulin, the platelet-derived growth factor,or a dystrophin protein. In order for the cell to produce themulti-subunit protein, a cell is infected with the recombinant viruscontaining each of the different subunits. Alternatively, differentsubunits of a protein may be encoded by the same transgene. In thiscase, a single transgene includes the DNA encoding each of the subunits,with the DNA for each subunit separated by an internal ribozyme entrysite (IRES). This is desirable when the size of the DNA encoding each ofthe subunits is small, e.g., the total size of the DNA encoding thesubunits and the IRES is less than five kilobases. As an alternative toan IRES, the DNA may be separated by sequences encoding a 2A peptide,which self-cleaves in a post-translational event. See, e.g., M. L.Donnelly, et al, J. Gen. Viral., 78(Pt 1):13-21 (Jan 1997); Furler, S.,et al, Gene Ther., 8(11):864-873 (June 2001); Klump H., et al., GeneTher., 8(10):811-817 (May 2001). This 2A peptide is significantlysmaller than an IRES, making it well suited for use when space is alimiting factor. However, the selected transgene may encode anybiologically active product or other product, e.g., a product desirablefor study.

Suitable transgenes may be readily selected by one of skill in the art.The selection of the transgene is not considered to be a limitation ofthis embodiment.

2. Regulatory Elements

In addition to the major elements identified above for the minigene, thevector also includes conventional control elements necessary which areoperably linked to the transgene in a manner that permits itstranscription, translation and/or expression in a cell transfected withthe plasmid vector or infected with the virus produced by the methodsdescribed herein and/or known to those of skill in the art. A “controlelement” or “control sequence” is a nucleotide sequence involved in aninteraction of molecules that contributes to the functional regulationof a polynucleotide, including replication, duplication, transcription,splicing, translation, or degradation of the polynucleotide. Theregulation can affect the frequency, speed, or specificity of theprocess, and can be enhancing or inhibitory in nature. Control elementsare known in the art, and examples of some are discussed elsewhere inthis specification. As used herein, “operably linked” sequences includeboth expression control sequences that are contiguous with the gene ofinterest and expression control sequences that act in trans or at adistance to control the gene of interest.

Expression control sequences include appropriate transcriptioninitiation, termination, promoter and enhancer sequences; efficient RNAprocessing signals such as splicing and polyadenylation (polyA) signals;sequences that stabilize cytoplasmic mRNA; sequences that enhancetranslation efficiency (i.e., Kozak consensus sequence); sequences thatenhance protein stability; and when desired, sequences that enhancesecretion of the encoded product.

A great number of expression control sequences, including promoterswhich are native, constitutive, inducible and/or tissue-specific, areknown in the art and may be utilized. Examples of constitutive promotersinclude, without limitation, the retroviral Rous sarcoma virus (RSV) LTRpromoter (optionally with the RSV enhancer), the cytomegalovirus (CMV)promoter (optionally with the CMV enhancer) [see, e.g., Boshart et al,Cell, 41:521-530 (1985)], the SV40 promoter, the dihydrofolate reductasepromoter, the β-actin promoter, the phosphoglycerol kinase (PGK)promoter, and the EF1α promoter [Invitrogen].

Inducible promoters allow regulation of gene expression and can beregulated by exogenously supplied compounds, environmental factors suchas temperature, or the presence of a specific physiological state, e.g.,acute phase, a particular differentiation state of the cell, or inreplicating cells only. Inducible promoters and inducible systems areavailable from a variety of commercial sources, including, withoutlimitation, Invitrogen, Clontech and Ariad. Many other systems have beendescribed and can be readily selected by one of skill in the art. Forexample, inducible promoters include the zinc-inducible sheepmetallothionine (MT) promoter and the dexamethasone (Dex)-induciblemouse mammary tumor virus (MMTV) promoter. Other inducible systemsinclude the T7 polymerase promoter system [International Publication No.WO 98/10088]; the ecdysone insect promoter [No et al, Proc. Natl. Acad.Sci. USA, 93:3346-3351 (1996)], the tetracycline-repressible system[Gossen et al, Proc. Natl. Acad. Sci. USA, 89:5547-5551 (1992)], thetetracycline-inducible system [Gossen et al, Science, 268:1766-1769(1995), see also Harvey et al, Curr. Opin. Chem. Biol., 2:512-518(1998)]. Other systems include the FK506 dimer, VP16 or p65 usingcastradiol, diphenol murislerone, the RU486-inducible system [Wang etal, Nat. Biotech., 15:239-243 (1997) and Wang et al, Gene Ther.,4:432-441 (1997)] and the rapamycin-inducible system [Magari et al, J.Clin. Invest., 100:2865-2872 (1997)]. The effectiveness of someinducible promoters increases over time. In such cases one can enhancethe effectiveness of such systems by inserting multiple repressors intandem, e.g., TetR linked to a TetR by an IRES. Alternatively, one canwait at least 3 days before screening for the desired function. One canenhance expression of desired proteins by known means to enhance theeffectiveness of this system. For example, using the Woodchuck HepatitisVirus Post-transcriptional Regulatory Element (WPRE).

In another embodiment, the native promoter for the transgene will beused. The native promoter may be preferred when it is desired thatexpression of the transgene should mimic the native expression. Thenative promoter may be used when expression of the transgene must beregulated temporally or developmentally, or in a tissue-specific manner,or in response to specific transcriptional stimuli. In a furtherembodiment, other native expression control elements, such as enhancerelements, polyadenylation sites or Kozak consensus sequences may also beused to mimic the native expression.

Another embodiment of the transgene includes a transgene operably linkedto a tissue-specific promoter. For instance, if expression in skeletalmuscle is desired, a promoter active in muscle should be used. Theseinclude the promoters from genes encoding skeletal β-actin, myosin lightchain 2A, dystrophin, muscle creatine kinase, as well as syntheticmuscle promoters with activities higher than naturally occurringpromoters (see Li et al., Nat. Biotech., 17:241-245 (1999)). Examples ofpromoters that are tissue-specific are known for liver (albumin,Miyatake et al., J. Virol., 71:5124-32 (1997); hepatitis B virus corepromoter, Sandig et al., Gene Ther., 3:1002-9 (1996); alpha-fetoprotein(AFP), Arbuthnot et al., Hum. Gene Ther., 7:1503-14 (1996)), boneosteocalcin (Stein et al., Mol. Biol. Rep., 24:185-96 (1997)); bonesialoprotein (Chen et al., J. Bone Miner. Res., 11:654-64 (1996)),lymphocytes (CD2, Hansal et al., J. Immunol., 161:1063-8 (1998);immunoglobulin heavy chain; T cell receptor chain), neuronal such asneuron-specific enolase (NSE) promoter (Andersen et al., Cell. Mol.Neurobiol., 13:503-15 (1993)), neurofilament light-chain gene (Piccioliet al., Proc. Natl. Acad. Sci. USA, 88:5611-5 (1991)), and theneuron-specific vgf gene (Piccioli et al., Neuron, 15:373-84 (1995)),among others.

Optionally, vectors carrying transgenes encoding therapeutically usefulor immunogenic products may also include selectable markers or reportergenes may include sequences encoding geneticin, hygromicin or purimycinresistance, among others. Such selectable reporters or marker genes(preferably located outside the viral genome to be packaged into a viralparticle) can be used to signal the presence of the plasmids inbacterial cells, such as ampicillin resistance. Other components of thevector may include an origin of replication. Selection of these andother promoters and vector elements are conventional and many suchsequences are available [see, e.g., Sambrook et al, and references citedtherein].

These vectors are generated using the techniques and sequences providedherein, in conjunction with techniques known to those of skill in theart. Such techniques include conventional cloning techniques of cDNAsuch as those described in texts [Sambrook et al, Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y.],use of overlapping oligonucleotide sequences of the adenovirus genomes,polymerase chain reaction, and any suitable method which provides thedesired nucleotide sequence.

III. Production of the Viral Vector

In one embodiment, the simian adenoviral plasmids (or other vectors) areused to produce adenoviral vectors. In one embodiment, the adenoviralvectors are adenoviral particles which are replication—defective. In oneembodiment, the adenoviral particles are rendered replication-defectiveby deletions in the E1a and/or E1b genes. Alternatively, theadenoviruses are rendered replication-defective by another means,optionally while retaining the E1a and/or E1b genes. The adenoviralvectors can also contain other mutations to the adenoviral genome, e.g.,temperature-sensitive mutations or deletions in other genes. In otherembodiments, it is desirable to retain an intact E1a and/or E1b regionin the adenoviral vectors. Such an intact E1 region may be located inits native location in the adenoviral genome or placed in the site of adeletion in the native adenoviral genome (e.g., in the E3 region).

In the construction of useful simian adenovirus vectors for delivery ofa gene to the human (or other mammalian) cell, a range of adenovirusnucleic acid sequences can be employed in the vectors. For example, allor a portion of the adenovirus delayed early gene E3 may be eliminatedfrom the simian adenovirus sequence which forms a part of therecombinant virus. The function of simian E3 is believed to beirrelevant to the function and production of the recombinant virusparticle. Simian adenovirus vectors may also be constructed having adeletion of at least the ORF6 region of the E4 gene, and more desirablybecause of the redundancy in the function of this region, the entire E4region. Still another SAdV-43, -45, -46, -47, -48, -49 or -50 vectorcontains a deletion in the delayed early gene E2a. Deletions may also bemade in any of the late genes L1 through L5 of the simian adenovirusgenome. Similarly, deletions in the intermediate genes IX and IVa₂ maybe useful for some purposes. Other deletions may be made in the otherstructural or non-structural adenovirus genes. The above discusseddeletions may be used individually, i.e., an adenovirus sequence for useas described herein may contain deletions in only a single region.Alternatively, deletions of entire genes or portions thereof effectiveto destroy their biological activity may be used in any combination. Forexample, in one exemplary vector, the adenovirus sequence may havedeletions of the E1 genes and the E4 gene, or of the E1, E2a and E3genes, or of the E1 and E3 genes, or of E1, E2a and E4 genes, with orwithout deletion of E3, and so on. As discussed above, such deletionsmay be used in combination with other mutations, such astemperature-sensitive mutations, to achieve a desired result.

An adenoviral vector lacking any essential adenoviral sequences (e.g.,E1a, E1b, E2a, E2b, E4 ORF6, L1, L2, L3, L4 and L5) may be cultured inthe presence of the missing adenoviral gene products which are requiredfor viral infectivity and propagation of an adenoviral particle. Thesehelper functions may be provided by culturing the adenoviral vector inthe presence of one or more helper constructs (e.g., a plasmid or virus)or a packaging host cell. See, for example, the techniques described forpreparation of a “minimal” human Ad vector in International PatentApplication WO96/13597, published May 9, 1996, and incorporated hereinby reference.

1. Helper Viruses

Thus, depending upon the simian adenovirus gene content of the viralvectors employed to carry the minigene, a helper adenovirus ornon-replicating virus fragment may be necessary to provide sufficientsimian adenovirus gene sequences necessary to produce an infectiverecombinant viral particle containing the minigene. Useful helperviruses contain selected adenovirus gene sequences not present in theadenovirus vector construct and/or not expressed by the packaging cellline in which the vector is transfected. In one embodiment, the helpervirus is replication-defective and contains a variety of adenovirusgenes in addition to the sequences described above. Such a helper virusis desirably used in combination with an E1-expressing cell line.

Helper viruses may also be formed into poly-cation conjugates asdescribed in Wu et al, J. Biol. Chem., 264:16985-16987 (1989); K. J.Fisher and J. M. Wilson, Biochem. J., 299:49 (Apr. 1, 1994). Helpervirus may optionally contain a second reporter minigene. A number ofsuch reporter genes are known to the art. The presence of a reportergene on the helper virus which is different from the transgene on theadenovirus vector allows both the Ad vector and the helper virus to beindependently monitored. This second reporter is used to enableseparation between the resulting recombinant virus and the helper virusupon purification.

2. Complementation Cell Lines

To generate recombinant simian adenoviruses (Ad) deleted in any of thegenes described above, the function of the deleted gene region, ifessential to the replication and infectivity of the virus, must besupplied to the recombinant virus by a helper virus or cell line, i.e.,a complementation or packaging cell line. In many circumstances, a cellline expressing the human E1 can be used to transcomplement the simianAd vector. This is particularly advantageous because, due to thediversity between the SAdV-43, -45, -46, -47, -48, -49 and -50 sequencesand the human AdE1 sequences found in currently available packagingcells, the use of the current human E1-containing cells prevents thegeneration of replication-competent adenoviruses during the replicationand production process. However, in certain circumstances, it will bedesirable to utilize a cell line which expresses the E1 gene productscan be utilized for production of an E1-deleted simian adenovirus. Suchcell lines have been described. See, e.g., U.S. Pat. 6,083,716.

If desired, one may utilize the sequences provided herein to generate apackaging cell or cell line that expresses, at a minimum, the adenovirusE1 gene from SAdV-43, -45, -46, -47, -48, -49 or -50 under thetranscriptional control of a promoter for expression in a selectedparent cell line. Inducible or constitutive promoters may be employedfor this purpose. Examples of such promoters are described in detailelsewhere in this specification. A parent cell is selected for thegeneration of a novel cell line expressing any desired SAdV-43, -45,-46, -47, -48, -49 or -50 gene. Without limitation, such a parent cellline may be HeLa [ATCC Accession No. CCL 2], A549 [ATCC Accession No.CCL 185], HEK 293, KB [CCL 17], Detroit [e.g., Detroit 510, CCL 72] andWI-38 [CCL 75] cells, among others. These cell lines are all availablefrom the American Type Culture Collection, 10801 University Boulevard,Manassas, Va. 20110-2209. Other suitable parent cell lines may beobtained from other sources.

Such E1-expressing cell lines are useful in the generation ofrecombinant simian adenovirus E1 deleted vectors. Additionally, oralternatively, cell lines that express one or more simian adenoviralgene products, e.g., E1a, E1b, E2a, and/or E4 ORF6, can be constructedusing essentially the same procedures are used in the generation ofrecombinant simian viral vectors. Such cell lines can be utilized totranscomplement adenovirus vectors deleted in the essential genes thatencode those products, or to provide helper functions necessary forpackaging of a helper-dependent virus (e.g., adeno-associated virus).The preparation of a host cell involves techniques such as assembly ofselected DNA sequences. This assembly may be accomplished utilizingconventional techniques. Such techniques include cDNA and genomiccloning, which are well known and are described in Sambrook et al.,cited above, use of overlapping oligonucleotide sequences of theadenovirus genomes, combined with polymerase chain reaction, syntheticmethods, and any other suitable methods which provide the desirednucleotide sequence.

In still another alternative, the essential adenoviral gene products areprovided in trans by the adenoviral vector and/or helper virus. In suchan instance, a suitable host cell can be selected from any biologicalorganism, including prokaryotic (e.g., bacterial) cells, and eukaryoticcells, including, insect cells, yeast cells and mammalian cells.Particularly desirable host cells are selected from among any mammalianspecies, including, without limitation, cells such as A549, WEHI, 3T3,10T1/2, HEK 293 cells or PERC6 (both of which express functionaladenoviral E1) [Fallaux, FJ et al, (1998), Hum Gene Ther, 9:1909-1917],Saos, C2C12, L cells, HT1080, HepG2 and primary fibroblast, hepatocyteand myoblast cells derived from mammals including human, monkey, mouse,rat, rabbit, and hamster. The selection of the mammalian speciesproviding the cells is not a limitation of this invention; nor is thetype of mammalian cell, i.e., fibroblast, hepatocyte, tumor cell, etc.

3. Assembly of Viral Particle and Transfection of a Cell Line

Generally, when delivering the vector comprising the minigene bytransfection, the vector is delivered in an amount from about 5 μg toabout 100 μg DNA, and preferably about 10 to about 50 μg DNA to about1×10⁴ cells to about 1×10¹³ cells, and preferably about 10⁵ cells.However, the relative amounts of vector DNA to host cells may beadjusted, taking into consideration such factors as the selected vector,the delivery method and the host cells selected.

The vector may be any vector known in the art or disclosed above,including naked DNA, a plasmid, phage, transposon, cosmids, episomes,viruses, etc. Introduction into the host cell of the vector may beachieved by any means known in the art or as disclosed above, includingtransfection, and infection. One or more of the adenoviral genes may bestably integrated into the genome of the host cell, stably expressed asepisomes, or expressed transiently. The gene products may all beexpressed transiently, on an episome or stably integrated, or some ofthe gene products may be expressed stably while others are expressedtransiently. Furthermore, the promoters for each of the adenoviral genesmay be selected independently from a constitutive promoter, an induciblepromoter or a native adenoviral promoter. The promoters may be regulatedby a specific physiological state of the organism or cell (i.e., by thedifferentiation state or in replicating or quiescent cells) or byexogenously-added factors, for example.

Introduction of the molecules (as plasmids or viruses) into the hostcell may also be accomplished using techniques known to the skilledartisan and as discussed throughout the specification. In preferredembodiment, standard transfection techniques are used, e.g., CaPO₄transfection or electroporation.

Assembly of the selected DNA sequences of the adenovirus (as well as thetransgene and other vector elements into various intermediate plasmids,and the use of the plasmids and vectors to produce a recombinant viralparticle are all achieved using conventional techniques. Such techniquesinclude conventional cloning techniques of cDNA such as those describedin texts [Sambrook et al, cited above], use of overlappingoligonucleotide sequences of the adenovirus genomes, polymerase chainreaction, and any suitable method which provides the desired nucleotidesequence. Standard transfection and co-transfection techniques areemployed, e.g., CaPO₄ precipitation techniques. Other conventionalmethods employed include homologous recombination of the viral genomes,plaquing of viruses in agar overlay, methods of measuring signalgeneration, and the like.

For example, following the construction and assembly of the desiredminigene-containing viral vector, the vector is transfected in vitro inthe presence of a helper virus into the packaging cell line. Homologousrecombination occurs between the helper and the vector sequences, whichpermits the adenovirus-transgene sequences in the vector to bereplicated and packaged into virion capsids, resulting in therecombinant viral vector particles. The current method for producingsuch virus particles is transfection-based. However, the invention isnot limited to such methods. The resulting recombinant simianadenoviruses are useful in transferring a selected transgene to aselected cell.

IV. Use of the Recombinant Adenovirus Vectors

The recombinant simian adenovirus (SAdV)-43, -45, -46, -47, -48, -49 or-50 based vectors are useful for gene transfer to a human or non-simianveterinary patient in vitro, ex vivo, and in vivo.

The recombinant adenovirus vectors described herein can be used asexpression vectors for the production of the products encoded by theheterologous genes in vitro. For example, the recombinant adenovirusescontaining a gene inserted into the location of an E1 deletion may betransfected into an E1-expressing cell line as described above.Alternatively, replication-competent adenoviruses may be used in anotherselected cell line. The transfected cells are then cultured in theconventional manner, allowing the recombinant adenovirus to express thegene product from the promoter. The gene product may then be recoveredfrom the culture medium by known conventional methods of proteinisolation and recovery from culture.

A SAdV-43, -45, -46, -47, -48, -49 or -50 -derived recombinant simianadenoviral vector provides an efficient gene transfer vehicle that candeliver a selected transgene to a selected host cell in vivo or ex vivoeven where the organism has neutralizing antibodies to one or more AAVserotypes. In one embodiment, the rAAV and the cells are mixed ex vivo;the infected cells are cultured using conventional methodologies; andthe transduced cells are re-infused into the patient. These compositionsare particularly well suited to gene delivery for therapeutic purposesand for immunization, including inducing protective immunity.

More commonly, the SAdV-43, -45, -46, -47, -48, -49 or -50 recombinantadenoviral vectors will be utilized for delivery of therapeutic orimmunogenic molecules, as described below. It will be readily understoodfor both applications, that the recombinant adenoviral vectors areparticularly well suited for use in regimens involving repeat deliveryof recombinant adenoviral vectors. Such regimens typically involvedelivery of a series of viral vectors in which the viral capsids arealternated. The viral capsids may be changed for each subsequentadministration, or after a pre-selected number of administrations of aparticular serotype capsid (e.g., one, two, three, four or more). Thus,a regimen may involve delivery of a rAd with a first simian capsid,delivery with a rAd with a second simian capsid, and delivery with athird simian capsid. A variety of other regimens which use the SAdV-43,-45, -46, -47, -48, -49 or -50 capsids alone, in combination with oneanother, or in combination with other adenoviruses (which are preferablyimmunologically non-crossreactive) will be apparent to those of skill inthe art. Optionally, such a regimen may involve administration of rAdwith capsids of other non-human primate adenoviruses, humanadenoviruses, or artificial sequences such as are described herein. Eachphase of the regimen may involve administration of a series ofinjections (or other delivery routes) with a single Ad capsid followedby a series with another capsid from a different Ad source.Alternatively, the SAdV-43, -45, -46, -47, -48, -49 or -50 vectors maybe utilized in regimens involving other non-adenoviral-mediated deliverysystems, including other viral systems, non-viral delivery systems,protein, peptides, and other biologically active molecules.

The following sections will focus on exemplary molecules which may bedelivered via the SAdV-43, -45, -46, -47, -48, -49 and -50 vectors.

A. Ad-Mediated Delivery of Therapeutic Molecules

In one embodiment, the above-described recombinant vectors areadministered to humans according to published methods for gene therapy.A simian viral vector bearing the selected transgene may be administeredto a patient, preferably suspended in a biologically compatible solutionor pharmaceutically acceptable delivery vehicle. A suitable vehicleincludes sterile saline. Other aqueous and non-aqueous isotonic sterileinjection solutions and aqueous and non-aqueous sterile suspensionsknown to be pharmaceutically acceptable carriers and well known to thoseof skill in the art may be employed for this purpose.

The simian adenoviral vectors are administered in sufficient amounts totransduce the target cells and to provide sufficient levels of genetransfer and expression to provide a therapeutic benefit without undueadverse or with medically acceptable physiological effects, which can bedetermined by those skilled in the medical arts. Conventional andpharmaceutically acceptable routes of administration include, but arenot limited to, direct delivery to the retina and other intraoculardelivery methods, direct delivery to the liver, inhalation, intranasal,intravenous, intramuscular, intratracheal, subcutaneous, intradermal,rectal, oral and other parenteral routes of administration. Routes ofadministration may be combined, if desired, or adjusted depending uponthe transgene or the condition. The route of administration primarilywill depend on the nature of the condition being treated.

Dosages of the viral vector will depend primarily on factors such as thecondition being treated, the age, weight and health of the patient, andmay thus vary among patients. For example, a therapeutically effectiveadult human or veterinary dosage of the viral vector is generally in therange of from about 100 μL to about 100 mL of a carrier containingconcentrations of from about 1×10⁶ to about 1×10¹⁵ particles, about1×10¹¹ to 1×10¹³ particles, or about 1×10⁹ to 1×10¹² particles virus.Dosages will range depending upon the size of the animal and the routeof administration. For example, a suitable human or veterinary dosage(for about an 80 kg animal) for intramuscular injection is in the rangeof about 1×10⁹ to about 5×10¹² particles per mL, for a single site.Optionally, multiple sites of administration may be delivered. Inanother example, a suitable human or veterinary dosage may be in therange of about 1×10¹¹ to about 1×10¹⁵ particles for an oral formulation.One of skill in the art may adjust these doses, depending on the routeof administration, and the therapeutic or vaccinal application for whichthe recombinant vector is employed. The levels of expression of thetransgene, or for an immunogen, the level of circulating antibody, canbe monitored to determine the frequency of dosage administration. Yetother methods for determining the timing of frequency of administrationwill be readily apparent to one of skill in the art.

An optional method step involves the co-administration to the patient,either concurrently with, or before or after administration of the viralvector, of a suitable amount of a short acting immune modulator. Theselected immune modulator is defined herein as an agent capable ofinhibiting the formation of neutralizing antibodies directed against arecombinant SAdV-43, -45, -46, -47, -48, -49 or -50 vector or capable ofinhibiting cytolytic T lymphocyte (CTL) elimination of the vector. Theimmune modulator may interfere with the interactions between the Thelper subsets (T_(H1) or T_(H2)) and B cells to inhibit neutralizingantibody formation. Alternatively, the immune modulator may inhibit theinteraction between T_(H1) cells and CTLs to reduce the occurrence ofCTL elimination of the vector. A variety of useful immune modulators anddosages for use of same are disclosed, for example, in Yang et al., J.Viral., 70(9) (September 1996); International Patent Application No. WO96/12406, published May 2, 1996; and International Patent ApplicationNo. PCT/U.S.96/03035, all incorporated herein by reference.

1. Therapeutic Transgenes

Useful therapeutic products encoded by the transgene include hormonesand growth and differentiation factors including, without limitation,insulin, glucagon, growth hormone (GH), parathyroid hormone (PTH),growth hormone releasing factor (GRF), follicle stimulating hormone(FSH), luteinizing hormone (LH), human chorionic gonadotropin (hCG),vascular endothelial growth factor (VEGF), angiopoietins, angiostatin,granulocyte colony stimulating factor (GCSF), erythropoietin (EPO),connective tissue growth factor (CTGF), basic fibroblast growth factor(bFGF), acidic fibroblast growth factor (aFGF), epidermal growth factor(EGF), transforming growth factor (TGF), platelet-derived growth factor(PDGF), insulin growth factors I and II (IGF-I and IGF-II), any one ofthe transforming growth factor superfamily, including TGF, activins,inhibins, or any of the bone morphogenic proteins (BMP) BMPs 1-15, anyone of the heregluin/neuregulin/ARIA/neu differentiation factor (NDF)family of growth factors, nerve growth factor (NGF), brain-derivedneurotrophic factor (BDNF), neurotrophins NT-3 and NT-4/5, ciliaryneurotrophic factor (CNTF), glial cell line derived neurotrophic factor(GDNF), neurturin, agrin, any one of the family ofsemaphorins/collapsins, netrin-1 and netrin-2, hepatocyte growth factor(HGF), ephrins, noggin, sonic hedgehog and tyrosine hydroxylase.

Other useful transgene products include proteins that regulate theimmune system including, without limitation, cytokines and lymphokinessuch as thrombopoietin (TPO), interleukins (IL) IL-1 through IL-25(including, e.g., IL-2, IL-4, IL-12 and IL-18), monocyte chemoattractantprotein, leukemia inhibitory factor, granulocyte-macrophage colonystimulating factor, Fas ligand, tumor necrosis factors and, interferons,and, stem cell factor, flk-2/flt3 ligand. Gene products produced by theimmune system are also useful. These include, without limitation,immunoglobulins IgG, IgM, IgA, IgD and IgE, chimeric immunoglobulins,humanized antibodies, single chain antibodies, T cell receptors,chimeric T cell receptors, single chain T cell receptors, class I andclass II MHC molecules, as well as engineered immunoglobulins and MHCmolecules. Useful gene products also include complement regulatoryproteins such as complement regulatory proteins, membrane cofactorprotein (MCP), decay accelerating factor (DAF), CR1, CF2 and CD59.

Still other useful gene products include any one of the receptors forthe hormones, growth factors, cytokines, lymphokines, regulatoryproteins and immune system proteins. Also encompassed herein arereceptors for cholesterol regulation, including the low densitylipoprotein (LDL) receptor, high density lipoprotein (HDL) receptor, thevery low density lipoprotein (VLDL) receptor, and the scavengerreceptor. Gene products such as members of the steroid hormone receptorsuperfamily including glucocorticoid receptors and estrogen receptors,Vitamin D receptors and other nuclear receptors are also contemplated.In addition, useful gene products include transcription factors such asjun, fos, max, mad, serum response factor (SRF), AP-1, AP2, myb, MyoDand myogenin, ETS-box containing proteins, TFE3, E2F, ATF1, ATF2, ATF3,ATF4, ZFS, NFAT, CREB, HNF-4, C/EBP, SP1, CCAAT-box binding proteins,interferon regulation factor (IRF-1), Wilms tumor protein, ETS-bindingprotein, STAT, GATA-box binding proteins, e.g., GATA-3, and the forkheadfamily of winged helix proteins.

Other useful gene products include, carbamoyl synthetase I, ornithinetranscarbamylase, arginosuccinate synthetase, arginosuccinate lyase,arginase, fumarylacetacetate hydrolase, phenylalanine hydroxylase,alpha-1 antitrypsin, glucose-6-phosphatase, porphobilinogen deaminase,factor VIII, factor IX, cystathione beta-synthase, branched chainketoacid decarboxylase, albumin, isovaleryl-coA dehydrogenase, propionylCoA carboxylase, methyl malonyl CoA mutase, glutaryl CoA dehydrogenase,insulin, beta-glucosidase, pyruvate carboxylate, hepatic phosphorylase,phosphorylase kinase, glycine decarboxylase, H-protein, T-protein, acystic fibrosis transmembrane regulator (CFTR) sequence, and adystrophin cDNA sequence.

Other useful gene products include non-naturally occurring polypeptides,such as chimeric or hybrid polypeptides having a non-naturally occurringamino acid sequence containing insertions, deletions or amino acidsubstitutions. For example, single-chain engineered immunoglobulinscould be useful in certain immunocompromised patients. Other types ofnon-naturally occurring gene sequences include antisense molecules andcatalytic nucleic acids, such as ribozymes, which could be used toreduce overexpression of a target.

Reduction and/or modulation of expression of a gene are particularlydesirable for treatment of hyperproliferative conditions characterizedby hyperproliferating cells, as are cancers and psoriasis. Targetpolypeptides include those polypeptides which are produced exclusivelyor at higher levels in hyperproliferative cells as compared to normalcells. Target antigens include polypeptides encoded by oncogenes such asmyb, myc, fyn, and the translocation gene bcr/abl, ras, src, P53, neu,trk and EGRF. In addition to oncogene products as target antigens,target polypeptides for anti-cancer treatments and protective regimensinclude variable regions of antibodies made by B cell lymphomas andvariable regions of T cell receptors of T cell lymphomas which, in someembodiments, are also used as target antigens for autoimmune disease.Other tumor-associated polypeptides can be used as target polypeptidessuch as polypeptides which are found at higher levels in tumor cellsincluding the polypeptide recognized by monoclonal antibody 17-1A andfolate binding polypeptides.

Other suitable therapeutic polypeptides and proteins include those whichmay be useful for treating individuals suffering from autoimmunediseases and disorders by conferring a broad based protective immuneresponse against targets that are associated with autoimmunity includingcell receptors and cells which produce self-directed antibodies. T cellmediated autoimmune diseases include Rheumatoid arthritis (RA), multiplesclerosis (MS), Sjögren's syndrome, sarcoidosis, insulin dependentdiabetes mellitus (IDDM), autoimmune thyroiditis, reactive arthritis,ankylosing spondylitis, scleroderma, polymyositis, dermatomyositis,psoriasis, vasculitis, Wegener's granulomatosis, Crohn's disease andulcerative colitis. Each of these diseases is characterized by T cellreceptors (TCRs) that bind to endogenous antigens and initiate theinflammatory cascade associated with autoimmune diseases.

The simian adenoviral vectors based on the SAdV-43, -45, -46, -47, -48,-49 or -50 sequences herein are particularly well suited for therapeuticregimens in which multiple adenoviral-mediated deliveries of transgenesis desired, e.g., in regimens involving redelivery of the same transgeneor in combination regimens involving delivery of other transgenes. Suchregimens may involve administration of a SAdV-43, -45, -46, -47, -48,-49 or -50 simian adenoviral vector, followed by re-administration witha vector from the same serotype adenovirus. Particularly desirableregimens involve administration of a SAdV-43, -45, -46, -47, -48, -49 or-50 simian adenoviral vector in which the source of the adenoviralcapsid sequences of the vector delivered in the first administrationdiffers from the source of adenoviral capsid sequences of the viralvector utilized in one or more of the subsequent administrations. Forexample, a therapeutic regimen involves administration of a SAdV-43,-45, -46, -47, -48, -49 or -50 vector and repeat administration with oneor more adenoviral vectors of the same or different serotypes. Inanother example, a therapeutic regimen involves administration of anadenoviral vector followed by repeat administration with a SAdV-43, -45,-46, -47, -48, -49 or -50 vector which has a capsid that differs fromthe source of the capsid in the first delivered adenoviral vector, andoptionally further administration with another vector which is the sameor, preferably, differs from the source of the adenoviral capsid of thevector in the prior administration steps. These regimens are not limitedto delivery of adenoviral vectors constructed using the SAdV-43, -45,-46, -47, -48, -49 or -50 sequences. Rather, these regimens can readilyutilize vectors other adenoviral sequences, including, withoutlimitation, other simian adenoviral sequences, (e.g., Pan9 or C68, C1,etc), other non-human primate adenoviral sequences, or human adenoviralsequences, in combination with one or more of the SAdV-43, -45, -46,-47, -48, -49 or -50 vectors. Examples of such simian, other non-humanprimate and human adenoviral serotypes are discussed elsewhere in thisdocument. Further, these therapeutic regimens may involve eithersimultaneous or sequential delivery of SAdV-43, -45, -46, -47, -48, -49or -50 adenoviral vectors in combination with non-adenoviral vectors,non-viral vectors, and/or a variety of other therapeutically usefulcompounds or molecules. The use of the SAdV-43, -45, -46, -47, -48, -49and -50 vectors is not limited to these therapeutic regimens, a varietyof which will be readily apparent to one of skill in the art.

B. Ad-Mediated Delivery of Immunogenic Transgenes

The recombinant SAdV-43, -45, -46, -47, -48, -49 and -50 vectors mayalso be employed as immunogenic compositions. As used herein, animmunogenic composition is a composition to which a humoral (e.g.,antibody) or cellular (e.g., a cytotoxic T cell) response is mounted toa transgene product delivered by the immunogenic composition followingdelivery to a mammal, and preferably a primate. A recombinant simian Adcan contain in any of its adenovirus sequence deletions a gene encodinga desired immunogen. The simian adenovirus is likely to be better suitedfor use as a live recombinant virus vaccine in different animal speciescompared to an adenovirus of human origin, but is not limited to such ause. The recombinant adenoviruses can be used as prophylactic ortherapeutic vaccines against any pathogen for which the antigen(s)crucial for induction of an immune response and able to limit the spreadof the pathogen has been identified and for which the cDNA is available.Such vaccine (or other immunogenic) compositions are formulated in asuitable delivery vehicle, as described above. Generally, doses for theimmunogenic compositions are in the range defined above for therapeuticcompositions. The levels of immunity of the selected gene can bemonitored to determine the need, if any, for boosters. Following anassessment of antibody titers in the serum, optional boosterimmunizations may be desired.

Optionally, a vaccine composition of a SAdV-43, -45, -46, -47, -48, -49or -50 vector may be formulated to contain other components, including,e.g., adjuvants, stabilizers, pH adjusters, preservatives and the like.Such components are well known to those of skill in the vaccine art.Examples of suitable adjuvants include, without limitation, liposomes,alum, monophosphoryl lipid A, and any biologically active factor, suchas cytokine, an interleukin, a chemokine, a ligands, and optimallycombinations thereof Certain of these biologically active factors can beexpressed in vivo, e.g., via a plasmid or viral vector. For example,such an adjuvant can be administered with a priming DNA vaccine encodingan antigen to enhance the antigen-specific immune response compared withthe immune response generated upon priming with a DNA vaccine encodingthe antigen only.

The recombinant adenoviruses are administered in a “an immunogenicamount”, that is, an amount of recombinant adenovirus that is effectivein a route of administration to transfect the desired cells and providesufficient levels of expression of the selected gene to induce an immuneresponse. Where protective immunity is provided, the recombinantadenoviruses are considered to be vaccine compositions useful inpreventing infection and/or recurrent disease.

Alternatively, or in addition, the vectors may contain a transgeneencoding a peptide, polypeptide or protein which induces an immuneresponse to a selected immunogen. The recombinant SAdV-43, -45, -46,-47, -48, -49 and -50 vectors are expected to be highly efficacious atinducing cytolytic T cells and antibodies to the inserted heterologousantigenic protein expressed by the vector.

For example, immunogens may be selected from a variety of viralfamilies. Example of viral families against which an immune responsewould be desirable include, the picornavirus family, which includes thegenera rhinoviruses, which are responsible for about 50% of cases of thecommon cold; the genera enteroviruses, which include polioviruses,coxsackieviruses, echoviruses, and human enteroviruses such as hepatitisA virus; and the genera apthoviruses, which are responsible for foot andmouth diseases, primarily in non-human animals. Within the picornavirusfamily of viruses, target antigens include the VP1, VP2, VP3, VP4, andVPG. Another viral family includes the calcivirus family, whichencompasses the Norwalk group of viruses, which are an importantcausative agent of epidemic gastroenteritis. Still another viral familydesirable for use in targeting antigens for inducing immune responses inhumans and non-human animals is the togavirus family, which includes thegenera alphavirus, which include Sindbis viruses, RossRiver virus, andVenezuelan, Eastern & Western Equine encephalitis, and rubivirus,including Rubella virus. The flaviviridae family includes dengue, yellowfever, Japanese encephalitis, St. Louis encephalitis and tick borneencephalitis viruses. Other target antigens may be generated from theHepatitis C or the coronavirus family, which includes a number ofnon-human viruses such as infectious bronchitis virus (poultry), porcinetransmissible gastroenteric virus (pig), porcine hemagglutinatingencephalomyelitis virus (pig), feline infectious peritonitis virus(cats), feline enteric coronavirus (cat), canine coronavirus (dog), andhuman respiratory coronaviruses, which may cause the common cold and/ornon-A, B or C hepatitis. Within the coronavirus family, target antigensinclude the E1 (also called M or matrix protein), E2 (also called S orSpike protein), E3 (also called HE or hemagglutin-elterose) glycoprotein(not present in all coronaviruses), or N (nucleocapsid). Still otherantigens may be targeted against the rhabdovirus family, which includesthe genera vesiculovirus (e.g., Vesicular Stomatitis Virus), and thegeneral lyssavirus (e.g., rabies).

Within the rhabdovirus family, suitable antigens may be derived from theG protein or the N protein. The family filoviridae, which includeshemorrhagic fever viruses such as Marburg and Ebola virus, may be asuitable source of antigens. The paramyxovirus family includesparainfluenza Virus Type 1, parainfluenza Virus Type 3, bovineparainfluenza Virus Type 3, rubulavirus (mumps virus), parainfluenzaVirus Type 2, parainfluenza virus Type 4, Newcastle disease virus(chickens), rinderpest, morbillivirus, which includes measles and caninedistemper, and pneumovirus, which includes respiratory syncytial virus.The influenza virus is classified within the family orthomyxovirus andis a suitable source of antigen (e.g., the HA protein, the N1 protein).The bunyavirus family includes the genera bunyavirus (Californiaencephalitis, La Crosse), phlebovirus (Rift Valley Fever), hantavirus(puremala is a hemahagin fever virus), nairovirus (Nairobi sheepdisease) and various unassigned bungaviruses. The arenavirus familyprovides a source of antigens against LCM and Lassa fever virus. Thereovirus family includes the genera reovirus, rotavirus (which causesacute gastroenteritis in children), orbiviruses, and cultivirus(Colorado Tick fever, Lebombo (humans), equine encephalosis, bluetongue).

The retrovirus family includes the sub-family oncorivirinal whichencompasses such human and veterinary diseases as feline leukemia virus,HTLVI and HTLVII, lentivirinal (which includes human immunodeficiencyvirus (HIV), simian immunodeficiency virus (SIV), felineimmunodeficiency virus (FIV), equine infectious anemia virus, andspumavirinal). Among the lentiviruses, many suitable antigens have beendescribed and can readily be selected. Examples of suitable HIV and SIVantigens include, without limitation the gag, pol, Vif, Vpx, VPR, Env,Tat, Nef, and Rev proteins, as well as various fragments thereof. Forexample, suitable fragments of the Env protein may include any of itssubunits such as the gp120, gp160, gp41, or smaller fragments thereof,e.g., of at least about 8 amino acids in length. Similarly, fragments ofthe tat protein may be selected. [See, U.S. Pat. Nos. 5,891,994 and6,193,981.] See, also, the HIV and SIV proteins described in D. H.Barouch et al, J. Virol., 75(5):2462-2467 (March 2001), and R. R. Amara,et al, Science, 292:69-74 (6 April 2001). In another example, the HIVand/or SIV immunogenic proteins or peptides may be used to form fusionproteins or other immunogenic molecules. See, e.g., the HIV-1 Tat and/orNef fusion proteins and immunization regimens described in InternationalPublication No. WO 01/54719, published Aug. 2, 2001, and InternationalPublication No. WO 99/16884, published Apr. 8, 1999. The HIV and/or SIVimmunogenic proteins or peptides described herein are only exemplary. Inaddition, a variety of modifications to these proteins has beendescribed or could readily be made by one of skill in the art. See,e.g., the modified gag protein that is described in U.S. Pat. No.5,972,596. Further, any desired HIV and/or SIV immunogens may bedelivered alone or in combination. Such combinations may includeexpression from a single vector or from multiple vectors. Optionally,another combination may involve delivery of one or more expressedimmunogens with delivery of one or more of the immunogens in proteinform. Such combinations are discussed in more detail below.

The papovavirus family includes the sub-family polyomaviruses (BKU andJCU viruses) and the sub-family papillomavirus (associated with cancersor malignant progression of papilloma). The adenovirus family includesviruses (EX, AD7, ARD, O.B.) which cause respiratory disease and/orenteritis. The parvovirus family includes feline parvovirus (felineenteritis), feline panleucopeniavirus, canine parvovirus, and porcineparvovirus. The herpesvirus family includes the sub-familyalphaherpesvirinae, which encompasses the genera simplexvirus (HSVI,HSVII), varicellovirus (pseudorabies, varicella zoster) and thesub-family betaherpesvirinae, which includes the genera cytomegalovirus(HCMV, muromegalovirus) and the sub-family gammaherpesvirinae, whichincludes the genera lymphocryptovirus, EBV (Burkitts lymphoma),infectious rhinotracheitis, Marek's disease virus, and rhadinovirus. Thepoxvirus family includes the sub-family chordopoxvirinae, whichencompasses the genera orthopoxvirus (Variola (Smallpox) and Vaccinia(Cowpox)), parapoxvirus, avipoxvirus, capripoxvirus, leporipoxvirus,suipoxvirus, and the sub-family entomopoxvirinae. The hepadnavirusfamily includes the Hepatitis B virus. One unclassified virus which maybe suitable source of antigens is the Hepatitis delta virus. Still otherviral sources may include avian infectious bursal disease virus andporcine respiratory and reproductive syndrome virus. The alphavirusfamily includes equine arteritis virus and various Encephalitis viruses.

Immunogens which are useful to immunize a human or non-human animalagainst other pathogens include, e.g., bacteria, fungi, parasiticmicroorganisms or multicellular parasites which infect human andnon-human vertebrates, or from a cancer cell or tumor cell. Examples ofbacterial pathogens include pathogenic gram-positive cocci includepneumococci; staphylococci; and streptococci. Pathogenic gram-negativecocci include meningococcus; gonococcus. Pathogenic entericgram-negative bacilli include enterobacteriaceae; pseudomonas,acinetobacteria and eikenella; melioidosis; salmonella; shigella;haemophilus; moraxella; H. ducreyi (which causes chancroid); brucella;Franisella tularensis (which causes tularemia); yersinia (pasteurella);streptobacillus moniliformis and spirillum; Gram-positive bacilliinclude listeria monocytogenes; erysipelothrix rhusiopathiae;Corynebacterium diphtheria (diphtheria); cholera; B. anthracia(anthrax); donovanosis (granuloma inguinale); and bartonellosis.Diseases caused by pathogenic anaerobic bacteria include tetanus;botulism; other clostridia; tuberculosis; leprosy; and othermycobacteria. Pathogenic spirochetal diseases include syphilis;treponematoses: yaws, pinta and endemic syphilis; and leptospirosis.Other infections caused by higher pathogen bacteria and pathogenic fungiinclude actinomycosis; nocardiosis; cryptococcosis, blastomycosis,histoplasmosis and coccidioidomycosis; candidiasis, aspergillosis, andmucormycosis; sporotrichosis; paracoccidiodomycosis, petriellidiosis,torulopsosis, mycetoma and chromomycosis; and dermatophytosis.Rickettsial infections include Typhus fever, Rocky Mountain spottedfever, Q fever, and Rickettsialpox. Examples of mycoplasma andchlamydial infections include: mycoplasma pneumoniae; lymphogranulomavenereum; psittacosis; and perinatal chlamydial infections. Pathogeniceukaryotes encompass pathogenic protozoans and helminths and infectionsproduced thereby include: amebiasis; malaria; leishmaniasis;trypanosomiasis; toxoplasmosis; Pneumocystis carinii; Trichans;Toxoplasma gondii; babesiosis; giardiasis; trichinosis; filariasis;schistosomiasis; nematodes; trematodes or flukes; and cestode (tapeworm)infections.

Many of these organisms and/or toxins produced thereby have beenidentified by the Centers for Disease Control [(CDC), Department ofHeath and Human Services, USA], as agents which have potential for usein biological attacks. For example, some of these biological agents,include, Bacillus anthracia (anthrax), Clostridium botulinum and itstoxin (botulism), Yersinia pestis (plague), variola major (smallpox),Francisella tularensis (tularemia), and viral hemorrhagic fevers[filoviruses (e.g., Ebola, Marburg], and arenaviruses [e.g., Lassa,Machupo]), all of which are currently classified as Category A agents;Coxiella burnetti (Q fever); Brucella species (brucellosis),Burkholderia mallei (glanders), Burkholderia pseudomallei (meloidosis),Ricinus communis and its toxin (ricin toxin), Clostridium perfringensand its toxin (epsilon toxin), Staphylococcus species and their toxins(enterotoxin B), Chlamydia psittaci (psittacosis), water safety threats(e.g., Vibrio cholerae, Crytosporidium parvum), Typhus fever (Richettsiapowazekii), and viral encephalitis (alphaviruses, e.g., Venezuelanequine encephalitis; eastern equine encephalitis; western equineencephalitis); all of which are currently classified as Category Bagents; and Nipan virus and hantaviruses, which are currently classifiedas Category C agents. In addition, other organisms, which are soclassified or differently classified, may be identified and/or used forsuch a purpose in the future. It will be readily understood that theviral vectors and other constructs described herein are useful todeliver antigens from these organisms, viruses, their toxins or otherby-products, which will prevent and/or treat infection or other adversereactions with these biological agents.

Administration of the SAdV-43, -45, -46, -47, -48, -49 and -50 vectorsto deliver immunogens against the variable region of the T cells areanticipated to elicit an immune response including CTLs to eliminatethose T cells. In RA, several specific variable regions of TCRs whichare involved in the disease have been characterized. These TCRs includeV-3, V-14, V-17 and Vα-17. Thus, delivery of a nucleic acid sequencethat encodes at least one of these polypeptides will elicit an immuneresponse that will target T cells involved in RA. In MS, severalspecific variable regions of TCRs which are involved in the disease havebeen characterized. These TCRs include V-7 and Vα-10. Thus, delivery ofa nucleic acid sequence that encodes at least one of these polypeptideswill elicit an immune response that will target T cells involved in MS.In scleroderma, several specific variable regions of TCRs which areinvolved in the disease have been characterized. These TCRs include V-6,V-8, V-14 and Vα-16, Vα-3C, Vα-7, Vα-14, Vα-15, Vα-16, Vα-28 and Vα-12.Thus, delivery of a recombinant simian adenovirus that encodes at leastone of these polypeptides will elicit an immune response that willtarget T cells involved in scleroderma.

C. Ad-Mediated Delivery Methods

The therapeutic levels, or levels of immunity, of the selected gene canbe monitored to determine the need, if any, for boosters. Following anassessment of CD8+T cell response, or optionally, antibody titers, inthe serum, optional booster immunizations may be desired. Optionally,the recombinant SAdV-43, -45, -46, -47, -48, -49 and -50 vectors may bedelivered in a single administration or in various combination regimens,e.g., in combination with a regimen or course of treatment involvingother active ingredients or in a prime-boost regimen. A variety of suchregimens has been described in the art and may be readily selected.

For example, prime-boost regimens may involve the administration of aDNA (e.g., plasmid) based vector to prime the immune system to second,booster, administration with a traditional antigen, such as a protein ora recombinant virus carrying the sequences encoding such an antigen.See, e.g., International Publication No. WO 00/11140, published Mar. 2,2000, incorporated by reference. Alternatively, an immunization regimenmay involve the administration of a recombinant SAdV-43, -45, -46, -47,-48, -49 or -50 vector to boost the immune response to a vector (eitherviral or DNA-based) carrying an antigen, or a protein. In still anotheralternative, an immunization regimen involves administration of aprotein followed by booster with a vector encoding the antigen.

In one embodiment, a method of priming and boosting an immune responseto a selected antigen by delivering a plasmid DNA vector carrying saidantigen, followed by boosting with a recombinant SAdV-43, -45, -46, -47,-48, -49 or -50 vector is described. In one embodiment, the prime-boostregimen involves the expression of multiproteins from the prime and/orthe boost vehicle. See, e.g., R. R. Amara, Science, 292:69-74 (6 Apr.2001) which describes a multiprotein regimen for expression of proteinsubunits useful for generating an immune response against HIV and SIV.For example, a DNA prime may deliver the Gag, Pol, Vif, VPX and Vpr andEnv, Tat, and Rev from a single transcript. Alternatively, the SIV Gag,Pol and HIV-1 Env is delivered in a recombinant SAdV-43, -45, -46, -47,-48, -49 or -50 adenovirus construct. Still other regimens are describedin International Publication Nos. WO 99/16884 and WO 01/54719.

However, the prime-boost regimens are not limited to immunization forHIV or to delivery of these antigens. For example, priming may involvedelivering with a first SAdV-43, -45, -46, -47, -48, -49 or -50 vectorfollowed by boosting with a second Ad vector, or with a compositioncontaining the antigen itself in protein form. In one example, theprime-boost regimen can provide a protective immune response to thevirus, bacteria or other organism from which the antigen is derived. Inanother embodiment, the prime-boost regimen provides a therapeuticeffect that can be measured using convention assays for detection of thepresence of the condition for which therapy is being administered.

The priming composition may be administered at various sites in the bodyin a dose dependent manner, which depends on the antigen to which thedesired immune response is being targeted. The amount or situs ofinjection(s) or to pharmaceutical carrier is not a limitation. Rather,the regimen may involve a priming and/or boosting step, each of whichmay include a single dose or dosage that is administered hourly, daily,weekly or monthly, or yearly. As an example, the mammals may receive oneor two doses containing between about 10 μg to about 50 μg of plasmid incarrier. A desirable amount of a DNA composition ranges between about 1μg to about 10,000 μg of the DNA vector. Dosages may vary from about 1μg to 1000 μg DNA per kg of subject body weight. The amount or site ofdelivery is desirably selected based upon the identity and condition ofthe mammal.

The dosage unit of the vector suitable for delivery of the antigen tothe mammal is described herein. The vector is prepared foradministration by being suspended or dissolved in a pharmaceutically orphysiologically acceptable carrier such as isotonic saline; isotonicsalts solution or other formulations that will be apparent to thoseskilled in such administration. The appropriate carrier will be evidentto those skilled in the art and will depend in large part upon the routeof administration. The compositions described herein may be administeredto a mammal according to the routes described above, in a sustainedrelease formulation using a biodegradable biocompatible polymer, or byon-site delivery using micelles, gels and liposomes. Optionally, thepriming step also includes administering with the priming composition, asuitable amount of an adjuvant, such as are defined herein.

Preferably, a boosting composition is administered about 2 to about 27weeks after administering the priming composition to the mammaliansubject. The administration of the boosting composition is accomplishedusing an effective amount of a boosting composition containing orcapable of delivering the same antigen as administered by the primingDNA vaccine. The boosting composition may be composed of a recombinantviral vector derived from the same viral source (e.g., SAdV-43, -45,-46, -47, -48, -49 or -50 adenoviral sequences, respectively) or fromanother source. Alternatively, the “boosting composition” can be acomposition containing the same antigen as encoded in the priming DNAvaccine, but in the form of a protein or peptide, which compositioninduces an immune response in the host. In another embodiment, theboosting composition contains a DNA sequence encoding the antigen underthe control of a regulatory sequence directing its expression in amammalian cell, e.g., vectors such as well-known bacterial or viralvectors. The primary requirements of the boosting composition are thatthe antigen of the composition is the same antigen, or a cross-reactiveantigen, as that encoded by the priming composition.

In another embodiment, the SAdV-43, -45, -46, -47, -48, -49 and -50vectors are also well suited for use in a variety of other immunizationand therapeutic regimens. Such regimens may involve delivery of SAdV-43,-45, -46, -47, -48, -49 or -50 vectors simultaneously or sequentiallywith Ad vectors of different serotype capsids, regimens in whichSAdV-43, -45, -46, -47, -48, -49 or -50 vectors are deliveredsimultaneously or sequentially with non-Ad vectors, regimens in whichthe SAdV-43, -45, -46, -47, -48, -49 or -50 vectors are deliveredsimultaneously or sequentially with proteins, peptides, and/or otherbiologically useful therapeutic or immunogenic compounds. Such uses willbe readily apparent to one of skill in the art.

The following examples illustrate the cloning of SAdV-43, -45, -46, -47,-48, -49 and -50 and the construction of exemplary recombinant vectors.These examples are illustrative only, and do not limit the scope of thepresent invention.

EXAMPLE 1 Isolation of Simian Adenoviruses (SAdV-43, -45, -46, -47, -48,-49 and -50) and PCR Analysis

Stool samples were collected at the facilities that house the animalsand were suspended in Hanks' Balanced Salt solution, and sent toUniversity of Pennsylvania on ice. The particulates were removed bycentrifugation, and sterile filtered through 0.2 μm syringe filters. 100μl of each filtered sample was inoculated into A549 cells grown in Ham'sF12 with 10% FBS, 1% Penn-Strep and 50 μg/mlgentamicin. After about 1 to2 weeks in culture, visual CPE was obvious in cell cultures with severalof the inoculates. The presence of adenoviruses in the cultures wasconfirmed by PCR amplification of an internal 1.9 kb of the hexon—theregion encompassing the hypervariable regions and that is predominantlyresponsible for conferring serotype specificity.

The primer pair that was utilized for PCR was SEQ ID NO:32:CAGGATGCTTCGGAGTACCTGAG and SEQ ID NO: 33: TTGGCNGGDATDGGGTAVAGCATGTT.The sequence obtained from this region was used to make an initialdetermination of adenoviral species and novelty of the serotype.Adenoviral isolates were plaque purified on A549 cells, propagated tohigh titer and purified on cesium chloride gradients using standardprocedures.

Viral DNAs obtained from purified virus preparations were completelysequenced (Qiagen Genomics Services, Hilden, Germany). In addition, asensitive nested PCR specific for the DNA polymerase with outer primers,SEQ ID NO: 34: TGATGCGYTTCTTACCTYTGGTYTCCATGAG and SEQ ID NO: 35:AGTTYTACATGCTGGGCTCTTACCG, and inner primers, SEQ ID NO: 36:GTGACAAAGAGGCTGTCCGTGTCCCCGTA and SEQ ID NO: 37:TCACGTGGCCTACACTTACAAGCCAATCAC, was used as a specific diagnostic foradenoviral shedding.

EXAMPLE 2 Phylogenetic Analysis

Eight (8) genes to use for subsequent phylogenetic analysis: fiber,hexon, penton, protease, E1A, DNA-binding protein (DBP), polymerase,terminal protein precursor (pTP). The annotated simian adenovirus genomeSAdV-25.2 was used as a reference to find the coding sequences of thesegenes in unannotated (target) genomes. To detect a gene in a targetgenome, the following procedure was performed: 1) the focal gene wasextracted from the reference genome and aligned it to the target genome.This provided a rough location of the focal gene in the target genome;2) all ORFs in the target genome that overlapped with the aligned regionwere identified; 3) if the gene was known to have an intron (E1A,polymerase and pTP genes), all GT-AG intron start-stop signal pairs thatpreserved the length of the first exon up to 30% and the length of theintron up to 60% with respect to the reference gene were identified; allsuch potential introns were spliced out, generating a pool of potentialcoding sequences; 4) for each coding sequence from this pool, thecorresponding amino acid sequence was aligned with the reference aminoacid sequence; 5) the coding sequences were sorted according the theiralignment score. The coding sequence with the highest score wasconsidered to be the coding sequence for the focal gene in the targetgenome. All alignments were performed with the ClustalW v. 2.0.9software [Thompson et al., Nucleic Acids Res, 22: 4673-4680 (1994)].

From the resulting nucleotide sequence alignments for each gene, thephylogenetic trees were reconstructed using the PhyML software [Guindonand Gascuel, Syst Biol, 52: 696-704 (2003)] under the HKY85 model[Hasegawa et al., J Mol Evol, 22: 160-174 (1985)] with atransition/transversion ratio, fraction of invariable sites and thediscretized gamma distribution of rates across sites [Yang, J Mol Evol.,39: 306-314 (1994)]. To assess the reliability of the reconstructedphylogenies, 100 bootstrap reconstructions were performed for each gene.

In order to accurately estimate the evolutionary rates within the B, C,E and D clades, all adenoviruses isolated from Old World monkeys as wellas HAdV-40 and HAdV-12 isolates were removed. The remaining proteinsequences were aligned and back-translated to DNA using the PAL2NALsoftware [Suyama et al., Nucl Acids Res, 34: W609-612 (2006)]. Thephylogenies for this were generated with this subset of data under thesame model as before. Finally, the PAML software [Yang, Mol Biol Evol,24: 1586-1591 (2007)] under the branch-specific model was used toestimate the dn/ds ratios on different branches keeping the branchlengths fixed. Internal analysis of homology was done by SIMPLOT slidingwindow analysis (window:1000bp, step: 20bp) [Lole et al., J Virol, 73:152-160 (1999)].

At the conclusion of this analysis, the sequences now designated simianadenoviruses 43 and 45 and disclosed herein were determined to be insubfamily C. The sequences now designated simian adenoviruses 46 and 47and disclosed herein were determined to be in subfamily B.

The sequence now designated simian adenoviruses 48, 49 and 50 anddisclosed herein were determined to fall outside each of subfamily A, B,C, D and E. SAdV-48, -49 and -50 are therefore positioned in apreviously uncharacterized clade. SAdV-48 is perhaps related to SAdV-3and SAdV-6, both previously isolated from Rhesus macaque and also bothoutside of the subfamilies A, B, C, D and E. Certain genes of SAdV-49and SAdV-50 demonstrated homology with species A and F adenoviruses.

EXAMPLE 3 Analysis of Cytokine Release by Plasmacytoid Dendritic CellCytokine Release Assay

PBMCs were isolated from whole blood collected in EDTA orheparin-containing Vacutainer tubes after Ficoll (Amersham Bioscience)density-gradient centrifugation at 1000 g for 25 minutes. Cells werecollected from the interphase and washed with PBS. PBMCs were incubatedwith ACK lysing buffer to lyse red blood cells, washed and resuspendedin complete RPMI medium (Mediatech) containing 10% FBS, 2 mM glutamine,10 mM HEPES, 50 μg/ml Gentamycin sulfate and Penn/strep.

Plasmacytoid dendritic cells were isolated from human PBMCs obtainedfrom the immunology core, Center for AIDS Research (CFAR), University ofPennsylvania, using a plasmacytoid dendritic cell isolation kit(Miltenyi biotec).

Cells were infected with different adenoviruses at a MOI of 10,000 asindicated. 48 hrs later the supernatants were collected and assayed forthe induction of various cytokine and chemokines using a combination ofELISA (IFN-alpha, Pierce) and multiplex-cytokine analysis (interleukin-g[IL-6] and macrophage inflammatory protein 1α [MIP-1α](Millipore/Luminex). Due to the extremely low numbers of pDCs inperipheral blood, the data presented represents cells isolated frommultiple donors.

Statistical significance of appropriate datasets was established by atwo-tailed paired student t-test when p<0.01.

Intracellular Cytokine Staining (ICCS)

Measurement of cytokine production by isolated lymphocytes was performedby combined surface and intracellular staining with monoclonalantibodies and subsequent five-color flow cytometric analysis. 10⁶lymphocytes were incubated with 10¹⁰ particles of heat inactivatedSAdV-48 or 1 μg of HAdV-5 hexon peptide library in the presence ofpurified antibodies to CD49d and CD28 (BD Biosciences) and GolgiPlug (BDPharmigen) for 6 h. Cells were washed and stained with ECD-CD8 (BeckmanCoulter) and APC-CD4 (BD Pharmigen) anti-human antibodies. Cells werewashed, permeabilized with 250 μl of Cytofix/Cytoperm solution at 4° C.for 20 min, washed with Perm/Wash solution, and stained withanti-cytokine antibodies including FITC-TNF-α (BD Pharmigen), PE-IL2(Beckman Coulter) and PE-Cy7-IFN-γ (BD Pharmigen) at 4° C. for 30 minCells were washed, examined by flow cytometry and the data were analyzedusing FlowJo software (Treestar, Ore.).

There were remarkable similarities in the profile of cytokine/chemokineexpression with adenoviruses belonging to the same species group. Thespecies C viruses failed to elaborate significant quantities of anycytokine/chemokine while high levels of several cytokines/chemokineswere produced after exposure to species E viruses. Detectable but morevariable levels of cytokine were found after exposure to the species Bviruses. Within each species group there were no consistent differencesbetween human and non-human primate adenoviruses.

IFN-γ ELISPOT

PBMCs obtained from rhesus macaque from Covance (N=15) and Oregon (N=15)and cynomolgus macaques from our facility (N=10) were evaluated foradenovirus-specific T cells against a variety of antigens [HAdV-5(species C), SAdV-24 (species E), SAdV-32 (species B), three macaquederived viruses (SADV-48, SAdV-49, and SAdV-50) and HAdV-5 hexonpeptides] using an IFN-γ ELISPOT assay.

Multiscreen 96-well titration plates (Millipore) were coated overnightwith antibody to human (Clone DK-1, Mabtech) or monkey (clone GZ-4,Mabtech) IFN-γ in PBS. Plates were washed and then blocked for 1 hourwith complete medium (RPMI containing 10% FBS). Plates were washed withRPMI medium, and lymphocytes were seeded in 100 ml of complete medium at1 and 2×10⁵ cells per well. Stimulant peptide pools or viruses wereadded to each well to a final concentration of 2 μg/ml of each peptideor at the various concentrations of viruses as indicated in the text ina 100 μl of complete medium. Cells were incubated at 37° C. for 20 hunder 5% CO₂. Plates were washed (PBS with 0.05% Tween-20) and thenincubated with biotinylated antibody to human and monkey IFN-γ (cloneB6-1; Mabtech) diluted in wash buffer containing 2% FBS. Plates wereincubated for 2 h and then washed. Avidin horseradish peroxidase (VectorLaboratories) was added to each well and plates incubated for 1 hour.Plates were washed and spots were developed with AEC substrate (BDBiosciences). Spots were counted with an automated ELISPOT reader (AID).PHA (Sigma) and CEF peptide pool (Mabtech) were included as positivecontrols in each analysis. Only ELISPOT counts greater than 55 spotforming units (SFU)/10⁶ lymphocytes and with values three times over thebackground were considered positive.

The ELISPOT data from cells stimulated with SAdV-48 showed that cellsfrom most animals failed to respond to any adenovirus antigen evaluatedalthough there were some animals with low level responses.

Selected rhesus and cynomolgus macaques were necropsied and mononuclearcells harvested from peripheral blood and various sites from the gut(ileum, colon and rectum) were evaluated for the presence ofadenovirus-specific T cells using HAdV-5 and SAdV-48 as antigens. Animalwelfare considerations precluded similar studies in great apes.Adenovirus-specific T cells were detected throughout the gut whenSAdV-48 was used to stimulate the cells; they demonstrated a mixture ofcells with central memory and effector-memory phenotypes. Interestinglythe presence of adenovirus-specific T cells in the gut was not predictedbased on analysis of the corresponding PBMCs which were consistentlynegative or had very low responses. T cells generated to endogenousadenovirus infections in macaques were not effectively stimulated withHAdV-5 using cells from blood or the mucosa. Intracellular cytokinestaining (ICCS) of gut samples from several macaques for adenovirususing SAdV-48 as an antigen revealed a predominance of CD4 T cellsalthough the frequencies were low but positive for expression of IFN-γ,TNF-α and IL-2. Stool extracts were also assayed for the presence ofneutralizing antibodies to SAdV-35 (species B) and SAdV-40 (species C).Both the prevalence and titer were found to be consistently lower thanthose in serum.

EXAMPLE 4 The E3 Loci of Human Adenoviruses Contain Fewer and SmallerCR1 Open Reading Frames Compared to the Corresponding SimianAdenoviruses

Previous studies of human adenoviruses have shown that the E3 locusencodes proteins important in modulating host responses but notessential for replication. Variations in the structure and function ofE3 open reading frames may contribute to the marked differences in hostresponses that were observed between great apes and humans.

The E3 region of adenovirus can encode up to 9 open reading frames. TheSAdV-48 adenovirus contains only 6 open reading frames, the 12.4Kprotein, CR1-α, CR1-α, RID-α, RID-β and the 14.7 protein. The SAdV-49and SAdV-50 adenoviruses contain only 5 open reading frames, the 12.4Kprotein, CR1 (having the highest homology to CR1-beta genes from otheradenoviruses), RID-α, RID-β and the 14.7 protein.

The up to 9 open reading frames found in many adenoviruses are highlyconserved across human and great ape adenoviruses, including the lastthree open reading frames encoding the anti-apoptosis proteins RID-α,RID-β, and the 14.7K protein [Tollefson et al., Nature, 392: 726-730(1998)], the product of the first E3 open reading frame encoding a 12.5Kprotein of unknown function, and the gp 19k protein which is known todown-regulate MHC class I expression [Andersson et al., Cell, 43:215-222 (1985)].

The remaining E3 open reading frames numbering from two to four indifferent isolates, encode trans-membrane proteins of unknown functionthat contain a conserved domain—CR1 (pfam PF02440) and are designatedCR1-α, CR1-β, CR1-γ and CR1-δ respectively. Each species group of virusdemonstrated a characteristic structure of the CR1 open reading framesthat varied between different species group. However within a speciesgroup there were consistent differences between human and great apeisolates.

Species C great ape viruses all contained three CR1 orfs: CR1-α, CR1-β,and CR1-γ; human representatives of the species C family consistentlywere missing the CR1-γorf. Species B great ape viruses contained thefull spectrum of the 4 CR1s; human representatives of the species Bfamily were either missing CR1-δ (human B2) or contained a substantiallytruncated version of CR1-δ (human B1). Species E great ape viruses alsocontained all 4 CR1 orfs; the sole human representative of this family,HAdV-4 contained a substantially truncated version of CR1-γ.

The apparent degeneration of CR1 structure in human viruses as comparedto great ape viruses may impact on the ability of the virus to evadehost immune detection and elimination. The relative loss of E3 functionin human viruses may explain the more effective T cell response anddecreased shedding observed in humans.

This analysis of the CR1 family of open reading frames helps to furtherrefine possible scenarios related to the cross species transmission ofadenoviruses. The structure of the E3 locus across the phylogenetic treeis likely reflective of duplication events in a common ancestor ofspecies A, B, C, D, E and F viruses, followed by subsequent gene losses(e.g., in case of CR1-δ) particularly in serotypes that infect humans.This is most consistent with the transmission of adenovirus from apes tohumans. The other less likely explanation is that independent multipleduplications occurred within the B, C, and E species subsequent to theirdivergence.

EXAMPLE 5 Construction of E1-Deleted Adenoviral Vectors

Vectors are prepared by conventional techniques, e.g., as described inRoy, et al., Human Gene Therapy 15:519-530 (May 2004). For example, asubfamily B E1-deleted vector is prepared as described in InternationalPublication No. WO 2009/073103, published Jun. 11, 2009. Similarly, asubfamily C E1-deleted vector is prepared as described in InternationalPublication No. WO 2009/105084, published Aug. 27, 2009.

In order to construct E1-deleted adenoviral vectors expressing theinfluenza virus nucleoprotein, the nucleotide sequence encoding the H1N1influenza A virus NP (A/Puerto Rico/8/34/Mount Sinai, GenBank accessionnumber AF389119.1) was codon optimized and completely synthesized(Celtek Genes, Nashville, Tenn.). An expression cassette composed of thehuman cytomegalovirus early promoter, a synthetic intron (obtained fromthe plasmid pCI (Promega, Madison, Wis.), the codon optimized influenzaA NP coding sequence and the bovine growth hormone polyadenylationsignal was constructed. The plasmid pShuttle CMV PI FluA NP harbors theabove described expression cassette where it is flanked by therecognition sites for the rare-cutting restriction enzymes I-CeuI andPI-SceI (New England Biolabs) respectively. In order to create amolecular clone of an E1-deleted adenoviral vector, plasmid molecularclones of the E1-deleted adenoviruses were first created whererecognition sites for the rare-cutting restriction enzymes I-CeuI andPI-SceI have been inserted in place of an E1 deletion. The E1-deletedadenoviral plasmids were then digested with I-CeuI and PI-SceI and theexpression cassette (digested by the same enzymes) was ligated in. Theresulting adenoviral plasmid molecular clones were transfected into HEK293 cells to rescue recombinant adenoviral vector. Rescue followingtransfection was found to be facilitated by first releasing the linearadenoviral genome from the plasmid by restriction enzyme digestion. SeeRoy, et al., Vaccine 25:6845-6851 (August 2007); InternationalPublication Nos. WO 2009/073103, published Jun. 11, 2009; and WO2009/105084, published Aug. 27, 2009.

EXAMPLE 6 Adenoviral Influenza A Nucleoprotein Tetramer Vaccines

BALB/c mice were injected intramuscularly with 1×10¹¹ particle formingunits (PFU) of a simian adenovirus (SAdV) vector encoding an influenza Anucleoprotein (NP) tetramer according to the protocol of Roy, et al.,Vaccine 25:6845-6851 (August 2007). Delivery by SAdV-45 (subfamily C)and SAdV-46 (subfamily B) was assessed against human adenovirus (HAdV) 5(subfamily C). Kinetics of Ter+ CD8+ T cell responses to the NP tetramerand T cell phenotypes were determined at days 10 and 20 post-injection.Cytokine profiles were determined at day 10 post-injection.

Kinetics

At 10 days post-injection, ˜8% of Ter+CD8+ T cells were specific for theNP delivered by HAdV-5. ˜5.5% of Ter+CD8+ T cells were specific for theNP delivered by SAdV-46. ˜0.5% of Ter+CD8+ T cells were specific for theNP delivered by SAdV-45[Percentages ˜+/−0.5%]. At 20 dayspost-injection, Ter+CD8+ T cells specific for the NP delivered by HAdV-5had decreased to 5.5%, Ter+CD8+ T cells specific for the NP delivered bySAdV-46 had increased to 7%, and Ter+CD8+ T cells specific for the NPdelivered by SAdV-45 remained constant.

Memory Phenotype

Ter+CD8+ T cells were evaluated for phenotype. At 10 dayspost-injection, ˜7.5% of the T cells were specific for the NP deliveredby HAdV-5. ˜0.5% were central memory T cells (TCM), ˜3% were effectormemory T cells (TEM), and ˜4% were effector T cells (TE). At day 20,˜5.5% of the T cells were specific for the NP delivered by HAdV-5. ˜0.5%were TCM, ˜2.5% were TEM, and ˜2.5% were TE.

At 10 days post-injection, ˜4.5% of the T cells were specific for the NPdelivered by HAdV-46. ˜0.5% were TCM, ˜2% were TEM, and ˜2% were TE. Atday 20, ˜7% of the T cells were specific for the NP delivered byHAdV-46. ˜0.5% were TCM, ˜4% were TEM, and ˜2.5% were TE. [Percentages˜+/−0.5%].

Cytokine Profile

CD8+ T cells were evaluated for secreted cytokine profile at 10 dayspost-injection in spleen and lung tissues. The data is summarized in thefollowing table.

Cytokine Profiles-Spleen % cytokine secreting CD8+ T cells (~+/−0.5)HAdV-5 SAdV-46 Total 6 5 IFNγ+ TNFα− IL2− 1 0.5 IFNγ+ TNFα+ IL2− 4.5 4IFNγ+ TNFα+ IL2+ 0.5 0.5

Cytokine Profiles-Lung % cytokine secreting CD8+ T cells (~+/−0.5)HAdV-5 SAdV-46 Total 25 30 IFNγ+ TNFα− IL2− 7.5 15 IFNγ+ TNFα+ IL2− 1714.5 IFNγ+ TNFα+ IL2+ 0.5 0.5

Results for SAdV-45 showed no immune response. However, this wasbelieved to be associated with vector production, which is beingrepeated.

All documents recited above, the Sequence Listing, U.S. ProvisionalPatent Application Nos: 61/109,955; 61/109,958; 61/109,957; 61/110,028;61/109,979; 61/109,986; 61/109,997; all filed Oct. 31, 2008,International Patent Application No. PCT/U.S.2009/062548, filed Oct. 29,2009, U.S. patent application Ser. No. 13/126,557, filed May 10, 2011,and U.S. patent application Ser. No. 14/548,631, filed Nov. 20, 2014,are incorporated herein by reference. Numerous modifications andvariations are included in the scope of the above-identifiedspecification and are expected to be obvious to one of skill in the art.Such modifications and alterations to the compositions and processes,such as selections of different minigenes or selection or dosage of thevectors or immune modulators are believed to be within the scope of theclaims appended hereto.

SEQUENCE LISTING FREE TEXT

The following information is provided for sequences containing free textunder numeric identifier <223>.

SEQ ID NO: (containing free text) Free text under <223> 1 Simianadenovirus type 43 2 Synthetic Construct 3 Synthetic Construct 4Synthetic Construct 5 Synthetic Construct 6 Synthetic Construct 7Synthetic Construct 8 Synthetic Construct 9 Synthetic Construct 10Synthetic Construct 11 Synthetic Construct 12 Synthetic Construct 13Synthetic Construct 14 Synthetic Construct 15 Synthetic Construct 16Synthetic Construct 17 Synthetic Construct 18 Synthetic Construct 19Synthetic Construct 20 Synthetic Construct 21 Synthetic Construct 22Synthetic Construct 23 Synthetic Construct 24 Synthetic Construct 25Synthetic Construct 26 Synthetic Construct 27 Synthetic Construct 28Synthetic Construct 29 Synthetic Construct 30 Simian adenovirus type 4331 Synthetic Construct 32 primer based on Simian adenovirus 33 primerbased on Simian adenovirus 34 primer based on Simian adenovirus 35primer based on Simian adenovirus 36 primer based on Simian adenovirus37 primer based on Simian adenovirus 38 Simian adenovirus type 45 39Synthetic Construct 40 Synthetic Construct 41 Synthetic Construct 42Synthetic Construct 43 Synthetic Construct 44 Synthetic Construct 45Synthetic Construct 46 Synthetic Construct 47 Synthetic Construct 48Synthetic Construct 49 Synthetic Construct 50 Synthetic Construct 51Synthetic Construct 52 Synthetic Construct 53 Synthetic Construct 54Synthetic Construct 55 Synthetic Construct 56 Synthetic Construct 57Synthetic Construct 58 Synthetic Construct 59 Synthetic Construct 60Simian adenovirus type 45 61 Synthetic Construct 62 Synthetic Construct63 Synthetic Construct 64 Synthetic Construct 65 Simian adenovirus type45 66 Synthetic Construct 67 Simian adenovirus type 45 68 SyntheticConstruct 69 Simian adenovirus type 46 70 Synthetic Construct 71Synthetic Construct 72 Synthetic Construct 73 Synthetic Construct 74Synthetic Construct 75 Synthetic Construct 76 Synthetic Construct 77Synthetic Construct 78 Synthetic Construct 79 Synthetic Construct 80Synthetic Construct 81 Synthetic Construct 82 Synthetic Construct 83Synthetic Construct 84 Synthetic Construct 85 Synthetic Construct 86Synthetic Construct 87 Synthetic Construct 88 Synthetic Construct 89Synthetic Construct 90 Simian adenovirus type 46 91 Synthetic Construct92 Synthetic Construct 93 Synthetic Construct 94 Synthetic Construct 95Simian adenovirus type 46 96 Synthetic Construct 97 Simian adenovirustype 46 98 Synthetic Construct 99 Simian adenovirus type 47 100Synthetic Construct 101 Synthetic Construct 102 Synthetic Construct 103Synthetic Construct 104 Synthetic Construct 105 Synthetic Construct 106Synthetic Construct 107 Synthetic Construct 108 Synthetic Construct 109Synthetic Construct 110 Synthetic Construct 111 Synthetic Construct 112Synthetic Construct 113 Synthetic Construct 114 Synthetic Construct 115Synthetic Construct 116 Synthetic Construct 117 Synthetic Construct 118Synthetic Construct 119 Synthetic Construct 120 Simian adenovirus type47 121 Synthetic Construct 122 Synthetic Construct 123 SyntheticConstruct 124 Synthetic Construct 125 Simian adenovirus type 47 126Synthetic Construct 127 Simian adenovirus type 47 128 SyntheticConstruct 129 Simian adenovirus type 48 130 Synthetic Construct 131Synthetic Construct 132 Synthetic Construct 133 Synthetic Construct 134Synthetic Construct 135 Synthetic Construct 136 Synthetic Construct 137Synthetic Construct 138 Synthetic Construct 139 Synthetic Construct 140Synthetic Construct 141 Synthetic Construct 142 Synthetic Construct 143Synthetic Construct 144 Synthetic Construct 145 Synthetic Construct 146Synthetic Construct 147 Synthetic Construct 148 Simian adenovirus type48 149 Synthetic Construct 150 Synthetic Construct 151 SyntheticConstruct 152 Synthetic Construct 153 Simian adenovirus type 48 154Synthetic Construct 155 Simian adenovirus type 48 156 SyntheticConstruct 157 Simian adenovirus type 49 158 Synthetic Construct 159Synthetic Construct 160 Synthetic Construct 161 Synthetic Construct 162Synthetic Construct 163 Synthetic Construct 164 Synthetic Construct 165Synthetic Construct 166 Synthetic Construct 167 Synthetic Construct 168Synthetic Construct 169 Synthetic Construct 170 Synthetic Construct 171Synthetic Construct 172 Synthetic Construct 173 Synthetic Construct 174Synthetic Construct 175 Synthetic Construct 176 Simian adenovirus type49 177 Synthetic Construct 178 Synthetic Construct 179 SyntheticConstruct 180 Synthetic Construct 181 Simian adenovirus type 49 182Synthetic Construct 183 Simian adenovirus type 50 184 SyntheticConstruct 185 Synthetic Construct 186 Synthetic Construct 187 SyntheticConstruct 188 Synthetic Construct 189 Synthetic Construct 190 SyntheticConstruct 191 Synthetic Construct 192 Synthetic Construct 193 SyntheticConstruct 194 Synthetic Construct 195 Synthetic Construct 196 SyntheticConstruct 197 Synthetic Construct 198 Synthetic Construct 199 SyntheticConstruct 200 Synthetic Construct 201 Synthetic Construct 202 Simianadenovirus type 50 203 Synthetic Construct 204 Synthetic Construct 205Synthetic Construct 206 Synthetic Construct 207 Simian adenovirus type50 208 Synthetic Construct

The invention claimed is:
 1. A recombinant adenovirus having an adenovirus capsid comprising a hexon protein, a penton protein, and a fiber protein, wherein said hexon protein is a hexon protein of SAdV-45 comprising the amino acids 1 to 953 of SEQ ID NO: 48; said capsid encapsidating a heterologous nucleic acid comprising a gene operably linked to expression control sequences which direct at least one of transcription, translation, and expression of the gene in a host cell.
 2. The recombinant adenovirus according to claim 1, further comprising a 5′ and a 3′ adenovirus cis-element necessary for replication and encapsidation.
 3. The recombinant adenovirus according to claim 1, wherein said adenovirus lacks all or a part of the E1 gene.
 4. The recombinant adenovirus according to claim 3, wherein said adenovirus is replication—defective.
 5. The recombinant adenovirus according to claim 1, wherein said capsid is a hybrid capsid.
 6. The recombinant adenovirus according to claim 5, wherein the hybrid capsid comprises at least one capsid protein from an adenovirus selected from the group consisting of SAdV-46, SAdV-43, SAdV-47, SAdV-48, SAdV-49, and SAdV-50.
 7. The recombinant adenovirus according to claim 6, wherein the penton protein is selected from: a penton protein of SAdV-46 with the amino acids 1 to 563 of SEQ ID NO: 74; a penton protein of SAdV-43 with the amino acids 1 to 651 of SEQ ID NO: 6; a penton protein of SAdV-47 with the amino acids 1 to 580 of SEQ ID NO: 104; a penton protein of SAdV-48 with the amino acids 1 to 504 of SEQ ID NO: 134; a penton protein of SAdV-49 with the amino acids 1 to 511 of SEQ ID NO: 162; and a penton protein of SAdV-50 with the amino acids 1 to 511 of SEQ ID NO:
 188. 8. The recombinant adenovirus according to claim 6, wherein the fiber protein is selected from: a fiber protein of SAdV-43 with amino acids 1 to 599 of SEQ ID NO: 22; a fiber protein of SAdV-46 with amino acids 1 to 353 of SEQ ID NO: 89; a fiber protein of SAdV-47 with amino acids 1 to 322 of SEQ ID NO: 119; a fiber protein of SAdV-48 with amino acids 1 to 570 of SEQ ID NO: 147; a fiber protein of SAdV-49 with amino acids 1 to 521 of SEQ ID NO: 174; a fiber protein of SAdV-49 with amino acids 1 to 418 of SEQ ID NO: 175; a fiber protein of SAdV-50 with amino acids 1 to 521 of SEQ ID NO: 200; and a fiber protein of SAdV-50 with amino acids 1 to 418 of SEQ ID NO:
 201. 9. A composition comprising the recombinant adenovirus according to claim 1 in a pharmaceutically acceptable carrier.
 10. A recombinant adenovirus having a capsid comprising a fiber protein, a penton protein, and a hexon protein, said hexon protein comprising (a) a full-length of amino acids about 51 to 953 of SEQ ID NO: 48 or (b) the full length of amino acids 1 to about 903 of SEQ ID NO: 48, said adenovirus capsid encapsidating a gene heterologous to SAdV-45 operably linked to expression control sequences which direct at least one of transcription, translation, and expression of the gene in a host cell.
 11. The recombinant adenovirus according to claim 10, wherein the fiber protein is of an adenovirus selected from SAdV-46, SAdV-43, SAdV-45, SAdV-47, SAdV-48, SAdV-49, and SAdV-50.
 12. The recombinant adenovirus according to claim 10, wherein the penton protein is of an adenovirus selected from SAdV-46, SAdV-43, SAdV-45, SAdV-47, SAdV-48, SAdV-49, and SAdV-50.
 13. The recombinant adenovirus according to claim 10, wherein said adenovirus is a pseudotyped adenovirus comprising a 5′ and a 3′ adenovirus cis-element necessary for replication and encapsidation, said cis-elements comprising an adenovirus 5′ inverted terminal repeat and an adenovirus 3′ inverted terminal repeat.
 14. The recombinant adenovirus according to claim 10, wherein the recombinant adenovirus comprises one or more adenovirus genes.
 15. The recombinant adenovirus according to claim 10, wherein the recombinant adenovirus is replication-defective.
 16. The recombinant adenovirus according to claim 15, wherein the recombinant adenovirus comprises a deletion in the E1 region. 